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METHODS IN ENZYMOLOGY
Editors-in-Chief
JOHN N. ABELSON and MELVIN I. SIMON

Division of Biology
California Institute of Technology
Pasadena, California
ANNA MARIE PYLE

Departments of Molecular, Cellular and Developmental
Biology and Department of Chemistry Investigator
Howard Hughes Medical Institute
Yale University
Founding Editors
SIDNEY P. COLOWICK and NATHAN O. KAPLAN

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First edition 2014
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Notices
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Practitioners and researchers must always rely on their own experience and knowledge in
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the safety of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of
products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions, or ideas contained in the material herein.
ISBN: 978-0-12-417158-9
ISSN: 0076-6879
For information on all Academic Press publications

visit our website at store.elsevier.com
CONTRIBUTORS
Veronica G. Anania
Department of Protein Chemistry, Genentech Inc., South San Francisco, California, USA
David Andrews
Department of Biological Sciences, Sunnybrook Research Institute, Toronto, Canada
Avi Ashkenazi
Cancer Immunology, Genentech, Inc., San Francisco, California, USA
Gregory H. Bird
Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology,
Dana-Farber Cancer Institute, and Department of Pediatrics, Children’s Hospital Boston,
Harvard Medical School, Boston, Massachusetts, USA
Craig R. Braun
Dana-Farber Cancer Institute; Department of Pediatrics, Children’s Hospital Boston, and
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
Xiaoke Chi
Department of Biological Sciences, Sunnybrook Research Institute, Toronto, and
Department of Chemical Biology, McMaster University, Hamilton, Canada
Charles S. Craik
Department of Pharmaceutical Chemistry, and Graduate Program in Chemistry and
Chemical Biology, University of California, San Francisco, California, USA
Kevin Dagbay
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
Peter M. Eimon
Department of Electrical Engineering and Computer Science, Massachusetts Institute of
Technology (MIT), Cambridge, Massachusetts, USA
Scott J. Eron
Jeanne A. Hardy
Mark G. Hinds
School of Chemistry, and Bio21 Molecular Science and Biotechnology Institute,
The University of Melbourne, Parkville, Victoria, Australia
Bradley T. Hyman
MassGeneral Institute for Neurodegenerative Disease, Department of Neurology,
Alzheimer’s Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical
School, Charlestown, Massachusetts, USA
xi
xii Contributors
Olivier Julien
Department of Pharmaceutical Chemistry, University of California, San Francisco,
California, USA
Young-Wook Jun
Graduate Program in Chemistry and Chemical Biology, and Department of Otolaryngology,
University of California, San Francisco, California, USA
Justin Kale
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, and
Akiko Koto
Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of
Tokyo, Tokyo, Japan
Erina Kuranaga
Laboratory for Histogenetic Dynamics, RIKEN CDB, Kobe, Japan
Marc Kvansakul
La Trobe Institute for Medical Science, La Trobe University, Bundoora, Victoria, Australia
Brian Leber
Department of Biochemistry and Biomedical Sciences, and Department of Medicine,
McMaster University, Hamilton, Canada
Susan Lee
Jennie R. Lill
Di Lin*
Min Lu
Peter D. Mace
Biochemistry Department, University of Otago, Dunedin, New Zealand
Masayuki Miura
Tokyo, and CREST, JST, Tokyo, Japan
Charles W. Morgan
Department of Pharmaceutical Chemistry, and Graduate Group in Chemistry and Chemical
Biology, University of California, San Francisco, California, USA
*Present address: College of Pharmacy, Purdue University, West Lafayette, IN, USA.
Contributors xiii
Shigekazu Nagata
Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto,
Japan
Pradeep Nair
Yu-ichiro Nakajima
Stowers Institute for Medical Research, Kansas, Missouri, USA
Samantha B. Nicholls
MassGeneral Institute for Neurodegenerative Disease, Department of Neurology,
Alzheimer’s Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical
School, Charlestown, Massachusetts, USA
Sean Petersen
Victoria C. Pham
Qui T. Phung
Stefan J. Riedl
Program in Cell Death and Survival Networks, NCI Designated Cancer Center,
Sanford-Burnham Medical Research Institute, La Jolla, California, USA
Stéphane G. Rolland
LMU Biocenter, Department Biology II, Ludwig-Maximilians-University, Munich,
Germany
Guy S. Salvesen
Program in Cell Death and Survival Networks, NCI Designated Cancer Center,
Sanford-Burnham Medical Research Institute, La Jolla, California, USA
Julia E. Seaman
Department of Pharmaceutical Chemistry, University of California, San Francisco,
California, USA
Banyuhay P. Serrano
Nirao M. Shah
Department of Anatomy, University of California, San Francisco, California, USA
Jean Philippe Stephan
Protein Chemistry Department/Discovery Oncology Department, Genentech Inc., South
San Francisco, California, USA
Jun Suzuki
Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto,
Japan
xiv Contributors
Cheryl Tajon
Department of Pharmaceutical Chemistry, and Graduate Program in Chemistry and
Chemical Biology, University of California, San Francisco, California, USA
Kiwamu Takemoto
PRESTO, JST, Tokyo, and Department of Physiology, Graduate School of Medicine,
Yokohama City University, Yokohama, Japan
Elizabeth K. Unger
Department of Anatomy, and Program in Biomedical Sciences, University of California,
San Francisco, California, USA
Sravanti Vaidya*
Elih M. Velázquez-Delgado†
Loren D. Walensky
James A. Wells
Department of Pharmaceutical Chemistry, and Department of Cellular and Molecular
Pharmacology, University of California, San Francisco, California, USA
Arun P. Wiita
Department of Pharmaceutical Chemistry, and Department of Laboratory Medicine,
University of California, San Francisco, California, USA
Yoshifumi Yamaguchi
Tokyo, and PRESTO, JST, Tokyo, Japan
Yunlong Zhao
*Present address: Biotechnology Department, MS Ramaiah Institute of Technology, Bangalore, India.

†
Present address: Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis,
TN, USA.
PREFACE
Cell turnover is a fundamental feature of metazoan biology. Severe damage

to cellular integrity usually causes passive, nonregulated cell death. In con-
trast, more confined disruption can lead to more deliberate cell elimination,
through specific mechanisms of Regulated Cell Death. In these two vol-
umes of Methods in Enzymology, we aim to highlight the current molecular
understanding of the major processes of Regulated Cell Death and to illus-
trate basic and advanced methodologies to study them. Volume A focuses on
the most extensively studied mode of cell death—apoptosis. Volume
B covers several nonapoptotic mechanisms, including necroptotic and auto-
phagic cell death. In Volume A, Chapters 1–4 cover various aspects of the
cell-intrinsic apoptosis pathway, including the Bcl-2 protein family and
mitochondria. Chapters 5 and 6 discuss death receptors and the extrinsic
pathway. Chapters 8–14 cover caspases—the apoptotic protease machine.
Chapter 15 highlights how apoptotic cells are recognized and cleared by
other cells, while Chapter 16 features the zebrafish as a versatile genetic
model organism for studying apoptosis. We hope these chapters will be con-
ceptually informative and practically useful for readers interested in the cur-
rent understanding and key open questions in each area as well as in
experimental strategies and techniques to interrogate the many facets of
apoptotic cell death including signaling, execution, and regulation.
AVI ASHKENAZI
JUNYING YUAN
JAMES A. WELLS
xv
CHAPTER ONE
Examining the Molecular

Mechanism of Bcl-2 Family
Proteins at Membranes by
Fluorescence Spectroscopy
Justin Kale*,†, Xiaoke Chi†,{, Brian Leber*,}, David Andrews†,1
*Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada
†
{
Department of Chemical Biology, McMaster University, Hamilton, Canada
}
Department of Medicine, McMaster University, Hamilton, Canada
1
Corresponding author: e-mail address: [email protected]
Contents
1. Introduction 2
2. An In Vitro Fluorescence-Based Liposome System 3
2.1 Expression and purification of Bcl-2 family proteins 4
2.2 Site-specific protein labeling 7
2.3 Production of mitochondria-like liposomes 9
3. Membrane Permeabilization Assay 11
3.1 ANTS/DPX release assay 11
4. Fluorescence Resonance Energy Transfer 13
4.1 Detecting the interaction between two proteins using FRET 15
5. Tracking the Conformation Changes of a Protein 16
5.1 NBD-emission assay 17
6. Determining the Topology of Proteins within Membranes 19
6.1 Iodide quenching of NBD-labeled Bax 19
7. Conclusion 20
Acknowledgments 22
References 22
Abstract
The Bcl-2 family proteins control apoptosis by regulation of outer mitochondrial mem-
brane permeabilization. Studying the Bcl-2 family is particularly difficult because the
functional interactions that regulate apoptosis occur at or within intracellular mem-
branes. Compared to other biophysical methods, fluorescence spectroscopy is well
suited to study membrane-bound proteins as experiments can be performed with
intact membranes and at protein concentrations similar to those found in cells. For
these reasons, fluorescence spectroscopy has been particularly useful in studying the
Methods in Enzymology, Volume 544 # 2014 Elsevier Inc. 1

ISSN 0076-6879 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-417158-9.00001-7
2 Justin Kale et al.
regulation of membrane permeabilization by Bcl-2 family proteins. Here, we discuss four

fluorescence-based assays used to study protein dynamics at membranes, with a focus
on how these techniques can be used to study the Bcl-2 family proteins.
1. INTRODUCTION
The Bcl-2 family of proteins regulates permeabilization of the outer
mitochondrial membrane (OMM). In most cell types, once the OMM is
permeabilized, the cell is committed to undergoing programmed cell death
(Budd, Tenneti, Lishnak, & Lipton, 2000). The sequence of events leading
to permeabilization of the OMM begins with prodeath signals triggering
posttranslational modifications of activator BH3-only proteins, such as the
cleavage of Bid to cBid (comprised of a p7 and p15 fragment, the latter also
referred to as tBid), that target them to the OMM where they bind to and
activate the pore-forming proteins Bax and Bak. Activation of Bax and Bak
results in their oligomerization within the OMM followed by
permeabilization of the OMM and release of intermembrane space proteins
such as cytochrome c and SMAC that act in downstream apoptotic path-
ways, culminating in cellular apoptosis (Shamas-Din, Kale, Leber, &
Andrews, 2013). The antiapoptotic proteins, such as Bcl-2 and Bcl-XL,
inhibit apoptosis by binding to and sequestering both BH3-only activators
and Bax/Bak (Bogner, Leber, & Andrews, 2010).
Significant research has been focused on determining the structure of
Bcl-2 family proteins. X-ray crystallography and nuclear magnetic reso-
nance (NMR) spectroscopy have revealed that the Bcl-2 family proteins
share a highly conserved core structure (Petros, Olejniczak, & Fesik,
2004). These studies have provided insight into how the Bcl-2 family pro-
teins bind to each other and suggest how they may interact with membranes.
However, the current relatively static structures for the Bcl-2 family are for
proteins without the lipid bilayer required for functional interactions of sev-
eral of the Bcl-2 family proteins (Leber, Lin, & Andrews, 2007). Determin-
ing the structures of proteins within a membrane mimetic environment
using X-ray crystallography and NMR spectroscopy is particularly difficult.
These techniques require a large amount of protein in a sample environment
that mimics but differs significantly from that of the cell and typically
includes detergents that can alter the functions of the Bcl-2 family proteins
(Hsu & Youle, 1997). As an example, unlike native Bax, detergent-treated
Fluorescence-Based Methods to Study the Bcl-2 Family Proteins 3
Bax can cause permeabilization of the OMM when added to isolated mito-
chondria (Antonsson, Montessuit, Lauper, Eskes, & Martinou, 2000), can
form oligomers that can be cross-linked in the absence of membranes
(Zhang et al., 2010) and has undergone a conformational change that is a
prerequisite for Bax activation (Yethon, Epand, Leber, Epand, &
Andrews, 2003). Additionally, the zwitterionic detergent CHAPS can pre-
vent the authentic interaction of Bax and tBid (Lovell et al., 2008), further
reinforcing the need to study the Bcl-2 family proteins in the absence of
detergents.
Fluorescence-based techniques are well suited to study protein dynamics
at membranes under physiological conditions in the absence of detergents
(Kale, Liu, Leber, & Andrews, 2012). Fluorescence spectroscopy allows
observation of protein:protein- and protein:membrane-binding dynamics
in real time, while gathering information about the kinetics and affinities
of these interactions that cannot be measured using typical structural tech-
niques due to complications from the membrane (Perez-Lara, Egea-
Jimenez, Ausili, Corbalan-Garcia, & Gomez-Fernandez, 2012; Satsoura
et al., 2012). Additionally, by using an environment-sensitive probe, it is
possible to determine the environment of specific residues as they undergo
conformational changes within membranes (Malhotra, Sathappa, Landin,
Johnson, & Alder, 2013; Shamas-Din, Bindner, et al., 2013). Initial
fluorescence-based studies of the Bcl-2 family proteins have used a simple
in vitro system to study the dynamic interactions that occur at, on, and within
membranes.
2. AN IN VITRO FLUORESCENCE-BASED LIPOSOME

SYSTEM
The functional interactions of the Bcl-2 family proteins occur in
membranes. Interaction of cBid with the membrane causes the p7 and
p15 fragments of cBid to dissociate, whereupon the p15 fragment (tBid)
undergoes a conformational change, that does not occur in solution and per-
mits binding between tBid and Bax within the membrane (Shamas-Din,
Bindner, et al., 2013; Shamas-Din, Kale, et al., 2013). Binding between
Bax and cBid or Bax and Bcl-XL requires membranes for an interaction
to occur as an interaction is not detected in solution (Billen, Kokoski,
Lovell, Leber, & Andrews, 2008; Lovell et al., 2008). Therefore, to study
the function of these proteins, a biochemical system is required that includes
a phospholipid bilayer that separates two distinct aqueous compartments
mimicking that of the cytoplasm and the interior of cellular organelles. We

and others (Bleicken et al., 2010; Landeta et al., 2011; Ren et al., 2010;
Shamas-Din, Bindner, et al., 2013; Shamas-Din, Kale, et al., 2013) have
used different variations of liposome or proteoliposome-based systems to
study the core mechanism of Bcl-2 family protein regulation of membrane
permeabilization. All of these systems lack the detergents typically required
for biochemical and structural studies of membrane proteins. For our studies,
fluorescently labeled purified full-length recombinant proteins and artificial
membranes in the form of liposomes with a composition mimicking that of
the OMM are used. This system is free of any other complicating factors
such as unknown binding partners that may be present at the OMM or
within the cytoplasm.
To use fluorescence to study proteins at membranes, it is essential to
make judicious choices of fluorophore, type of measurement, and instru-
ment. Fluorescence measurements require excitation of the fluorophore
by illuminating the sample with a specific wavelength of light and then
recording the emission from the fluorophore. Upon excitation, after some
period of time, termed the fluorescence lifetime (typically 1–10 ns), the fluo-
rophore returns to the ground electronic state via emission of a photon at a
lower energy, and thus longer wavelength, than the illuminating (excitation)
light. Because the emitted fluorescence is of much lower intensity than the
excitation light, the system must be free of molecules that absorb the emitted
light, and fluorescence contaminants that may interfere with the emission
signal from the fluorophore. Molecules such as quenchers that provoke non-
radiative decay of the fluorophore must also be avoided as they change the
fluorescence properties of the dyes. If these conditions are met, changes in
both fluorescence lifetime and emission intensity can provide specific infor-
mation about the underlying biochemical properties of the protein the fluo-
rophore is attached to (Lakowicz, 2006).
2.1. Expression and purification of Bcl-2 family proteins

2.1.1 Expression of Bax, Bcl-XL, and Bid
1. Escherichia coli are transformed (BL21-AI, New England Biolabs for cBid
and Bax; DH5a, New England Biolabs, for Bcl-XL) with the full-length
Bax, Bcl-XL, or Bid expression plasmid, plated on LB-ampicillin agar,
and then incubated overnight at 37 C. Bax and Bcl-XL are expressed
with a carboxy-terminal intein-chitin binding domain (IMPACT
expression system, New England Biolabs) and Bid is expressed with
an amino terminal 6 histidine tag.
2. The next day, a single colony is picked and used to inoculate 100 mL of
LB-ampicillin and grown overnight at 30 C with shaking. Then 1–3 L
of LB-ampicillin is inoculated with the overnight culture (20 mL for
each liter of LB-ampicillin) and grown at 37 C with shaking until
the bacterial growth is in log phase (OD600 is typically between 0.6
and 0.8) at which point protein expression is induced with either arab-
inose (0.2%, w/v, BL21-AI cells) or IPTG (1 mM, DH5a cells). Bacteria
are then incubated for 3–5 h at 30 C with shaking, harvested using cen-
trifugation, and stored at 20 C. We find that, for both Bax and Bcl-
XL, a longer expression time (5 h) yields more recombinant protein.
2.1.2 Purification of Bax and Bcl-XL

1. The bacterial pellet is resuspended in either Bax or Bcl-XL lysis buffer
(10 mL for each 2.5 g of bacterial pellet) and lysed via French press.
Lysed cells are centrifuged at 20,000 g and the cell lysate is incubated
with 1.5 mL of chitin bead slurry (New England Biolabs) for 2 h at 4 C
while rotating.
2. The cell lysate and resin slurry is then loaded into an Econo-Pac chro-
matography column (BioRad, Cat. #: 732-1010EDU) and the lysate
passed through (three to four times) before the resin is washed with
50 mL of either Bax or Bcl-XL wash buffer. The column is equilibrated
with either Bax or Bcl-XL cleavage buffer (10 mL) and capped with
1 mL of cleavage buffer remaining on top of the chitin beads followed
by incubation for 24–36 h at 4 C. The cleavage buffer contains hydrox-
ylamine, which causes cleavage of the intein-chitin binding domain all-
owing full-length Bax and Bcl-XL to be eluted. Bax or Bcl-XL is then
eluted with cleavage buffer (4 1 mL fractions). The majority of the
protein is typically within fractions 1 and 2.
3. a. Bax: A 0.2-mL bed volume DEAE–Sepharose column is prepared
and equilibrated with 2.5 mL of Bax-cleavage buffer (without
hydroxylamine). Bax elution fraction 1 and 2 are pooled and passed
through the column three times which removes additional contam-
inants that bind to the column.
b. Bcl-XL: A 0.3-mL bed volume high-performance phenyl Sepharose
column is equilibrated with 3 mL of Bcl-XL wash buffer. Bcl-XL
elution fractions 1–4 are pooled and applied to the column where
Bcl-XL binds and the column is washed with 5 mL of Bcl-XL wash
buffer (no PMSF in the buffer is needed). Bcl-XL is eluted (3 1 mL
fractions) with Bcl-XL wash buffer that does not contain NaCl
or PMSF.
4. Both Bax and Bcl-XL are dialyzed against 3 1 L of dialysis buffer (4 C
with stirring). After dialysis, the protein can be aliquoted and stored at
80 C or can be labeled with fluorescent dyes (see Section 2.2).
2.1.3 Purification of cBid

1. The bacterial pellet is resuspended in Bid-lysis buffer (10 mL for each
2.5 g of bacterial pellet) and lysed via French Press. Lysed cells are cen-
trifuged at 20,000 g, and the cell lysate is incubated with 0.8 mL
Ni-NTA agarose slurry (Qiagen) for 1.5 h at 4 C while rotating.
2. The cell lysate and resin slurry is then added to a Poly-Prep column
(BioRad, Cat. #: #731-1550EDU) and the lysate passed through three
times. The column is washed with 50 mL of Bid-wash buffer, and Bid is
eluted with 10 mL of Bid-elution buffer (collecting 5 1 mL fractions).
The first two fractions typically contain the highest concentration of Bid
and are pooled together. At this point, Bid can be cleaved to cBid (see
below, step 3), or if labeling Bid with a fluorescent dye, the pooled frac-
tions are first dialyzed 3 1 L against dialysis buffer at 4 C with stirring
and then labeled (see Section 2.2), followed by Bid cleavage and a final
dialysis step.
3. To produce cBid, the pooled Bid elutions are adjusted to contain 40 mM
HEPES, 1 mM EDTA, 10 mM DTT and incubated with 500 U of
recombinant human caspase-8 (Enzo Life Sciences, Cat. #: BML-
SE172-5000), and incubated for 48 h with rotating in the dark at room
temperature. The cBid sample is next dialyzed against 3 1 L of dialysis
buffer (4 C with stirring) and then aliquoted and stored at 80 C.
2.1.4 Buffer recipes

2.1.4.1 Bax
Bax-lysis buffer: 10 mM HEPES pH 7.0, 100 mM NaCl, 0.2% (w/v)
CHAPS, 1 mM PMSF, DNase, RNase
Bax-wash buffer: 10 mM HEPES pH 7.0, 500 mM NaCl, 0.5% (w/v)
CHAPS
Bax-cleavage buffer: 10 mM HEPES pH 7.0, 200 mM NaCl, 0.1% (w/v)
CHAPS, 100 mM Hydroxylamine. It is important to check the pH of
the cleavage buffer after adding hydroxylamine as it decreases the pH,
which will lead to insufficient yield of protein.
2.1.4.2 Bcl-XL
Bcl-XL-lysis buffer: 20 mM Tris pH 8.0, 500 mM NaCl, 1% (w/v)
CHAPS, 1 mM PMSF, DNase, RNase
Bcl-XL-wash buffer: 20 mM Tris pH 8.0, 200 mM NaCl, 0.2% (w/v)
CHAPS, 20% (v/v) glycerol, 1 mM PMSF
Bcl-XL-cleavage buffer: 20 mM Tris pH 8.0, 200 mM NaCl, 0.2% (w/v)
CHAPS, 20% (v/v) glycerol, 1 mM PMSF, 100 mM Hydroxylamine.
It is important to check the pH of the cleavage buffer after adding
hydroxylamine as it decreases the pH, which will lead to insufficient
yield of protein.
2.1.4.3 Bid
Bid-lysis buffer: 10 mM HEPES pH 7.0, 100 mM NaCl, 10 mM imidaz-
ole, 1 mM PMSF, DNase, RNase
Bid-wash buffer: 10 mM HEPES pH 7.0, 300 mM NaCl, 10 mM imidaz-
ole, 1% (w/v) CHAPS
Bid-elution buffer: 10 mM HEPES pH 7.0, 100 mM NaCl, 200 mM imid-
azole, 0.1% (w/v) CHAPS, 10% (v/v) glycerol
2.1.4.4 Dialysis
Dialysis buffer (for Bax, cBid, and Bcl-XL): 10 mM HEPES pH 7.0,
100 mM NaCl, 0.1 mM EDTA, 10% (v/v) glycerol
Extensive dialysis is needed to remove CHAPS which can alter the func-
tion and binding interactions of Bcl-2 family proteins. We typically dia-
lyze our purified protein samples for a minimum of 4 h against 1 L of
buffer, followed by dialysis overnight (12–16 h) against 1 L of buffer
and a final dialysis against 1 L of buffer for a minimum of 4 h. The
use of spin-concentrator columns should be avoided, in our experience,
as they severely attenuate the function of the Bcl-2 family proteins.
2.2. Site-specific protein labeling

The fluorescence-based techniques we use to study the Bcl-2 family require
purified recombinant proteins labeled with a fluorophore at a specific loca-
tion. There are two main options for labeling proteins, thiol or primary
amine labeling. Cysteine residues are less abundant than lysines in most pro-
tein sequences, thus we most frequently create single-cysteine mutants to
label the protein as this approach minimizes the number of mutations
required. There is a full spectrum of fluorescent probes available for pur-
chase, which have different spectral properties that can be ordered with
attached thiol reactive moieties such as iodoacetamide or maleimide deriv-

atives. Dyes must be chosen that are compatible not only with your protein
of choice but also with the system and equipment available.
In the methods reported below, the proteins were labeled with the low-
molecular weight fluorescent probes DAC (N-(7-dimethylamino-4-
methylcoumarin-3-yl) maleimide; Anaspec, Cat. #: 81403) and NBD
(N,N0 -dimethyl-N-(Iodoacetyl)-N0 -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
ethylenediamine; Molecular Probes, Cat. #: D-2004). The small size of these
dyes is a distinct advantage as they rarely perturb protein function; however,
measurements of NBD fluorescence require a sensitive instrument as the
quantum yield (ratio of photons emitted to photons absorbed) is low. More-
over, excitation of DAC requires an ultraviolet light source and both the exci-
tation and emission of this dye overlap endogenous fluorophores in cells
typically limiting its use to liposome-based systems. Many brighter (higher
quantum yields and extinction coefficients) fluorescent dyes have molecular
weights above 1 kDa, and in our experience these larger dyes frequently
change the function of the protein they are attached to.
Initially, it is best to follow the labeling protocol included by the man-
ufacturer when labeling your protein of interest; however, it is often neces-
sary to deviate from these conditions to get labeling that is both specific and
efficient.
1. For Bid, Bax, and Bcl-XL, the protein-labeling reaction is performed in
a HEPES-based buffer at pH 7.0–7.5 (10 mM HEPES, 200 mM
NaCl, 0.4%, w/v CHAPS). This pH range allows the cysteines to be
most-reactive while decreasing the reactivity of primary amines.
A 10–20 M excess of dye is added to the sample tube dropwise, to pre-
vent protein denaturation as dyes are typically dissolved in DMSO, and
the labeling reaction is rotated at room temperature for approximately
2 h in the dark. A reducing agent (5 mM DTT) is then added to quench
the reaction.
2. a. Bax and Bcl-XL: Free dye is removed via gel filtration over a G-25 fine
Sephadex column (10 mL bed volume, equilibrated with dialysis
buffer). Bax- and Bcl-XL-labeling reactions are applied to the column
and approximately 12 0.5 mL fractions are collected.
b. Bid: The Bid-labeling reaction is applied to a Ni-NTA column
(0.2 mL bed volume) and passed through three to four times all-
owing labeled Bid to bind to the column. The column is washed
with 50 mL of Bid-wash buffer and eluted with 5 mL of Bid-elution
buffer, collecting 4 0.5 mL fractions.
3. Absorbance spectroscopy is used to determine protein containing frac-

tions. Absorbance at both 280 nm (to detect protein) and at the absor-
bance peak of the dye used is determined. The fractions containing the
highest amount of protein are pooled. Bid can now be cleaved (see
Section 2.1.3, step 3). Bax, Bcl-XL, and cBid are then dialyzed against
3 1 L dialysis buffer at 4 C with stirring to remove any remaining free
dye and detergent, and the protein is aliquoted and stored for later use.
4. After dialysis, labeling efficiency is calculated by first determining the
concentration of your protein via absorbance at 280 (Bax), BCA assay
(Bcl-XL), or Bradford assay (cBid). Then the concentration of the dye
in the protein sample is determined by the OD of the sample at the peak
absorbance wavelength of the label, as outlined in the protocol supplied
by the manufacturer. Assuming that the protein is only labeled at the
single-cysteine residue and that there is no free dye in the sample, the
concentration of the dye should equal that of the labeled protein. Label-
ing efficiency is the fraction of labeled protein to that of total protein.
Single-cysteine mutants of the purified recombinant protein need to be
assayed functionally before and after labeling to determine if the mutation
or the addition of the dye alters protein function. Ideally, we begin using
mutants where one of the endogenous cysteines is present to minimize
the amount of mutations introduced into the protein. If the protein does
not contain any cysteines, choosing which residue to mutate to cysteine
for efficient labeling and proper protein function is largely empirical. Typ-
ically, if the structure is known, one begins using solvent exposed residues,
since those located in hydrophobic regions are difficult to label. Algorithms
used to predict solvent exposure or antigenicity (antigenic sites tend to be
both structured and solvent exposed) can often be useful in selecting a loca-
tion if the structure of your protein is unknown.
2.3. Production of mitochondria-like liposomes

Large unilamellar vesicles (LUVs) are liposomes that have with a mean diam-
eter of 120–140 nm and one lipid bilayer (Hope, Bally, Webb, & Cullis,
1985). OMM-like LUVs have been established as a valid biochemical model
for membrane permeablization by Bcl-2 family members (Kuwana et al.,
2002). These liposomes are assembled from lipids in fixed molar ratio similar
to that of the OMM, based upon lipid composition studies from solvent
extracted Xenopus mitochondria (Kuwana et al., 2002). Such liposome-
based systems allow the analysis of Bcl-2 family proteins in a simple context
while preserving their authentic functions. It is possible to more directly

explore Bcl-2 family function in this kind of system because the protein
and lipid components are well defined and tractable, unlike isolated mito-
chondria or proteoliposomes prepared from membranes.
2.3.1 Preparing lipid films and generating liposomes

1. Chloroform solublized lipids are added to a glass test tube to make a lipid
mixture of a defined composition (Table 1.1) to a total of 1 mg lipid
mass. The chloroform is evaporated off with nitrogen gas while rotating
the tube to ensure an even distribution of lipids on the wall and then put
under vacuum for 2 h at room temperature to remove any remaining
chloroform. The dry lipid film is then either used immediately or can
be stored for up to 2 weeks at 20 C. To reduce lipid oxidation by
atmospheric oxygen during storage, it is advisable to layer nitrogen or
argon gas on top of the lipid film and seal the tube with parafilm.
2. The dry 1 mg lipid film is hydrated with 1 mL of assay buffer (10 mM
HEPES, 200 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, pH 7). The lipids
become suspended and spontaneously form lipid bilayer vesicles due to
Table 1.1 Mitochondria-like lipid film composition

Amount
Molecular needed for
Molar weight 1 mg lipid
Name Company Catalog # (%) (g/mol) film (mg)
“PC”: L-a- Avanti 840051C 48 770.123 0.4596
phosphatidylcholine (egg,
chicken)
“PE”: L-a- Avanti 841118C 28 726.076 0.2528
phosphatidylethanolamine
(egg, chicken)
“PI”: L-a- Avanti 840042C 10 902.133 0.1122
phosphatidylinositol
(liver, bovine)
“DOPS”: 1,2-dioleoyl-sn- Avanti 840035C 10 810.025 0.1007
glycero-3-phospho-L-
serine
“TOCL”: 1,10 ,2,20 - Avanti 710335C 4 1501.959 0.0747
tetra-(9Z-octadecenoyl)
cardiolipin
the association of the hydrophobic tails, forming the center of the

bilayer, and the grouping of the hydrophilic heads of the phospholipids,
forming the edges of the bilayer. However, these vesicles are mul-
tilamellar as they contain more than one lipid bilayer and their size dis-
tribution is not homogeneous. To generate unilamellar liposomes, the
lipid mixture is subjected to 8–10 freeze/thaw cycles by alternately plac-
ing the sample vial in liquid nitrogen and a warm water bath (Hope et al.,
1985). The unilamellar liposomes are extruded 11 times through a filter
with 0.1 mm pore size to produce liposomes of a uniform size, at a final
concentration of 1 mg/mL lipid.
3. MEMBRANE PERMEABILIZATION ASSAY

The Bcl-2 family proteins play a pivotal role in regulating apoptosis by
controlling the permeabilization of the OMM through the activation of
Bax/Bak. Thus, a membrane permeablization assay is one crucial functional
assay for the Bcl-2 family proteins. To assay liposome permeabilization, the
liposomes are encapsulated with a polyanionic fluorophore, ANTS (8-
aminonaphthalene-1,3,6-trisulfonic acid; Molecular Probes, Cat. #:
A350), and cationic quencher, DPX (p-xylene-bis-pyridinium bromide;
Molecular Probes, Cat. #: X1525). Due to the high local concentration
of DPX, ANTS fluorescence is quenched when liposomes are still intact.
Recombinant Bax and/or other Bcl-2 family proteins and/or reagents are
added to the system in order to assay permeabilization. As the liposomes
permeabilize, ANTS and DPX are released from the liposomes, greatly
decreasing the local concentration of the quencher resulting in a gain of
ANTS fluorescence. The kinetics and extent of membrane permeabilization
can reveal crucial information for studying relationships between Bcl-2 fam-
ily members and how they regulate membrane permeabilization.
3.1. ANTS/DPX release assay

1. A dry 1 mg lipid film is hydrated with 1 mL of assay buffer with the addi-
tion of ANTS (12.5 mM) and DPX (45 mM). The lipid suspension is
vortexed until the ANTS and DPX dissolve, and liposomes are created
as above via 10 freeze/thaw cycles and extrusion through a 0.1 mm pore
size membrane.
2. Excess ANTS and DPX are removed by applying the extruded lipo-
somes onto a CL2B size-exclusion column (10 mL bed volume), that
separates the ANTS/DPX encapsulated liposomes from the free
ANTS/DPX in solution (Billen et al., 2008; Yethon et al., 2003). Frac-

tions (1 mL each) are collected in glass tubes and the liposome containing
fractions (typically fractions 3 and 4) are identified by an increase
in cloudiness of the sample which occurs due to light scattering by
the liposomes. The two liposome containing fractions are combined
resulting in a final ANTS/DPX liposome concentration of approxi-
mately 0.5 mg/mL lipid. These liposomes can now be used to test the
regulation of membrane permeabilization by the Bcl-2 family proteins.
3. The assay is set up in a low protein binding 96-well plate (Corning; Cat.
#: 3686) and in each well to be measured, 8 mL of ANTS/DPX lipo-
somes are added to 92 mL of assay buffer. Background measurements
(F0) are recorded at 30 C using a fluorescence plate reader (Tecan
M1000 pro) set to excite the sample at 355 nm (5 nm bandwidth) and
collect emission at 520 nm (12 nm bandwidth).
4. Proteins are added to the desired concentrations and combinations in
each well and fluorescence emission of ANTS (F) is recorded every
minute for 3 h at 30 C. Any increase in fluorescence emission is directly
related to membrane permeabilization.
5. To normalize the data, 100% ANTS release is determined by the addi-
tion of Triton to each well at a final concentration of 0.2% (w/v) causing
permeabilization of all liposomes and ANTS fluorescence is measured
(F100). This results in a slight overestimation of the intensity of 100%
release due to the dye becoming trapped in detergent micelles. Never-
theless, the release percentage generally does not take this into account
and is calculated as follows:
F F0
ANTS release ð%Þ ¼ 100%
F 100 F 0
The ANTS/DPX release assay can be used to dissect exactly how the dif-
ferent classes of Bcl-2 family proteins affect permeabilization of the
OMM. When cBid (20 nM), Bax (100 nM), or Bcl-XL (40 nM) are added
individually to liposomes they do not cause membrane permeabilization
(Fig. 1.1A). Incubation of liposomes with cBid and Bax results in membrane
permeabilization due to cBid binding to membranes causing separation of
the two fragments of cBid with the p15 (tBid) fragment remaining
membrane-bound and -activating Bax. Bcl-XL inhibits this process by bind-
ing to and inhibiting both tBid and Bax (Billen et al., 2008; Lovell et al.,
2008). Obviously, other techniques are needed to discern exactly how these
Figure 1.1 (A) Endpoint values of ANTS assay with 100 nM Bax, 20 nM cBid, 40 nM Bcl-XL
or both, or with 100 nM Bax, 20 nM tBid, and 40 nM Bcl-XL. (n ¼ 3). (B) Liposomes encap-
sulated with ANTS and DPX were incubated with 100 nM Bax, 20 nM cBid, or both. Mem-
brane permeabilization was assayed by an increase of ANTS fluorescence.
interactions occur (see Section 4); however, this dye release assay allows the
functional consequence of the addition of any number of various combina-
tions of Bcl-2 family members or small molecule effectors of the proteins to
be determined. Furthermore, it provides information on how changes in rel-
ative concentrations of the proteins can vary the extent of permeabilization
or how alterations in the parameters of the assay affect membrane
permeabilization. For example, it is possible quantify how changes in lipo-
some composition affect Bcl-2 family proteins functions to permeabilize
membranes or test specific mutations that may inhibit/activate the protein
of interest. Additionally, the kinetics of pore formation can be studied all-
owing the comparison of kinetics for Bax-mediated membrane
permeabilization in response to various BH3-only activators (Fig. 1.1B).
4. FLUORESCENCE RESONANCE ENERGY TRANSFER

Here, fluorescence resonance energy transfer (FRET) will be used to
detect binding between cBid and Bax, and Bax oligomerization. FRET is
possible between fluorophores when the emission spectra of one fluorescent
molecule, termed the donor, overlaps the excitation spectra of another fluo-
rophore, the acceptor. When a donor fluorophore is excited by light, an
electron moves to a higher energy state and, in the presence of an acceptor,
the energy is transferred nonradiatively to the acceptor fluorophore via
dipole–dipole interactions between the two probes. This transfer of energy
results in a decrease of the donor emission, and it is the change in the light
emitted by the donor that we track to measure FRET between two proteins.
One of the main advantages of FRET is that it requires both the donor
and acceptor fluorophores to be in close proximity for the required dipole
coupling to occur. As a result, FRET efficiency decreases to the sixth power
of distance according to the formula for FRET efficiency (E) at a fixed donor
acceptor distance:
R60
E¼
R60 + r 6
where R0 is the F€
orster distance, the distance between a donor acceptor pair
at which a 50% FRET efficiency is observed and r is the distance between the
donor and acceptor. The distance dependence of FRET is illustrated in
Fig. 1.2A where FRET efficiency is calculated for distances between a donor
Figure 1.2 (A) FRET efficiency as a function of distance between a dye pair with a the-
oretical Fo€rster distance of 50 Å. (B) FRET between cBid-DAC (20 nM) and Bax-NBD
(100 nM) in the presence (black circles) and absence (gray circles) of liposomes
(0.2 mg/mL). (C) FRET between Bax-DAC (20 nM) and Bax-NBD (100 nM) in samples con-
taining liposomes (0.2 mg/mL) with (black circles) or without (gray circles) 20 nM cBid.
and acceptor pair with an R0 of 50 Å (Lakowicz, 2006). For this dye pair,
FRET will only be detected if the distance between the two fluorophores
is 70 Å or less. Typical R0 values for a donor and acceptor pair are between
30 and 60 Å, similar to the size of proteins; thus, if FRET between donor-
and acceptor-labeled proteins is detected then they are bound to each other.
4.1. Detecting the interaction between two proteins

using FRET
As mentioned in Section 1, the BH3-only protein cBid targets to and
embeds within the OMM where it recruits and activates cytosolic Bax
(Leber et al., 2007; Lovell et al., 2008). Active membrane-bound Bax
oligomerizes within the OMM resulting in membrane permeabilization.
Here, we are using DAC and NBD as the donor and acceptor molecules,
respectively. We will be using FRET to detect (1) the binding between cBid
and Bax and (2) the binding between Bax molecules during oligomerization.
1. Liposomes are made as above (Section 2.3.1) resulting in liposomes at a
concentration of 1 mg/mL lipid.
2. The fluorimeter (Photon Technology International) is set to record the
fluorescence of DAC (380 nm excitation, 2 nm slit width; 460 nm emis-
sion, 10 nm slit width) with stirring for 1 h at 37 C. Either 200 mL of
liposomes and 800 mL of assay buffer, or as a control, 1 mL of assay buffer
is added to a quartz cuvette and the signal is read until it remains stable
(5 min). Two reactions are required to detect FRET. One that con-
tains both the donor- and acceptor-labeled proteins and a control that
contains the donor-labeled protein and unlabeled acceptor protein. This
control accounts for any changes in the donor protein that occur due to
binding interactions, conformational changes, or environment changes
that may affect the spectral properties of the donor dye.
3. The donor-labeled protein is added to the cuvette at a concentration of
20 nM and DAC fluorescence is read until the signal is stable. At this
point, the acceptor protein that is either labeled with NBD or unlabeled
is added to the system at a concentration of 100 nM. It is important to
keep the amount of acceptor higher (5–10) than that of the donor.
Keeping the donor protein in excess ensures, it will be saturated by
the acceptor.
4. The DAC signal is recorded for 1 h at 37 C. FRET efficiency (E) is
measured by comparing the relative intensity of the donor in the pres-
ence of labeled (FDA) and unlabeled (FD) acceptor and is calculated by:
F DA
E ¼1
FD
Figure 1.2 illustrates two binding interactions between the Bcl-2 family pro-
teins cBid and Bax. Donor (DAC)-labeled cBid (20 nM) is incubated with
acceptor (NBD)-labeled Bax (100 nM), and only in the presence of lipo-
somes do the two proteins interact (Fig. 1.2B). This underlines the point that
many functional interactions of the Bcl-2 family proteins only occur in the
presence of a lipid bilayer. Additionally, the activator protein cBid is
required for Bax to oligomerize, since FRET between donor (DAC) and
acceptor (NBD)-labeled Bax is only observed when cBid is added to the sys-
tem (Fig. 1.2C). As we observe the interactions of two proteins in real time,
kinetics of the reactions can be determined. Indeed, it is clear from the data
shown that the cBid–Bax interaction occurs faster than Bax oligomerization,
suggesting that cBid first binds to and activates Bax followed by Bax oligo-
merization. Additionally, it is possible to generate a binding curve where an
affinity for the interaction can be determined as was done for the binding
between cBid and Bax (Lovell et al., 2008). To do this multiple FRET mea-
surements are obtained by titrating the amount of acceptor, while keeping
the donor concentration fixed.
5. TRACKING THE CONFORMATION CHANGES

OF A PROTEIN
NBD is an environment-sensitive low-molecular weight fluorescent
dye that has been used to track environment changes of specific residues
of proteins (Dattelbaum et al., 2005; Lin, Jongsma, Pool, & Johnson,
2011). The emission intensity and fluorescence lifetime increases and the
emission peak of NBD blueshifts from 570 nm, in an aqueous environment,
to 530 nm when it is in a hydrophobic environment due to a decrease in
fluorescence quenching by water (Crowley, Reinhart, & Johnson, 1993).
The small size of NBD allows specific labeling of single-cysteine mutants
of proteins, with less potential perturbation of wild-type function. Impor-
tantly, NBD is uncharged but has sufficient polar characteristics that it
remains stable in both polar and nonpolar environments such that it is less
likely than other environment-sensitive dyes to change the membrane-
binding characteristics and/or conformation of the protein being studied
(Shepard et al., 1998). These properties of NBD make it particularly useful
to study membrane-binding proteins such as Bax and cBid that transition
from the aqueous environment and embed into a membrane bilayer (Lovell
et al., 2008; Shamas-Din, Bindner, et al., 2013; Shamas-Din, Kale,
et al., 2013).
5.1. NBD-emission assay

Real-time changes in the fluorescence of NBD can be measured to deter-
mine whether and when specific regions of Bax (labeled with NBD) insert
into the membrane during the activation of Bax. It is known from chemical-
labeling studies that Bax inserts helices 5, 6, and 9 into the membrane (Annis
et al., 2005). By labeling Bax at residue 175 (helix 9), it is possible to track the
conformational change of Bax as it transitions from a soluble monomer to
membrane embedded oligomer.
1. The fluorimeter is set to record NBD fluorescence (475 nm excitation,
2 nm slit width; 530 nm emission, 10 nm slit width), and as in the FRET
experiment above, 200 mL of 1 mg/mL liposomes are added to 800 mL
of assay buffer in a quartz cuvette. Background signal (Bg) is recorded
with stirring until stable at 37 C.
2. NBD-labeled Bax (100 nM) is added to the cuvette. Since Bax does not
insert into membranes in the absence of an activator (Hsu & Youle,
1998), Bax-NBD has a stable signal when incubated with liposomes
and an initial fluorescence value can be recorded (F0). Alternatively,
the very first point upon addition of the protein can be used as the F0
value if the protein insert into lipids too rapidly. This approach is useful
for proteins that are unstable in the assay solution such as cBid which
spontaneously targets to membranes (Shamas-Din, Bindner, et al.,
2013; Shamas-Din, Kale, et al., 2013). In the absence of membranes,
cBid has sufficient exposed hydrophobicity that it tends to aggregate
and to stick to the walls of the cuvette.
3. In our example, the change in emission over time (DF) of the dye labeled
Bax is collected once an activator, cBid, is added. Fluorescence intensity
plateaus after the protein comes to equilibrium (1 h endpoint). By cal-
culating the DF value, one can track the relative change in emission
intensity of the labeled residue in real time:
F Bg
△F ¼
F 0 Bg
Both residues 3 and 175 of Bax transition to a more hydrophobic environ-

ment as indicated (Fig. 1.3A) by the relative change in emission (DF). As the
environment change of the residue can be tracked over time, kinetics of

membrane binding can be measured. Tracking the kinetics of the environ-
ment changes of various residues as a protein undergoes a conformational
change has been used to order specific structural changes of the protein
(Shamas-Din, Bindner, et al., 2013; Shamas-Din, Kale, et al., 2013). Here,
the carboxyl terminus of Bax (residue 175) has slower kinetics compared to
that of the amino terminus of Bax (residue 3) suggesting that Bax undergoes
a conformational change at residue 3 before that of 175. Additionally, res-
idue 175 moves to a more hydrophobic environment since Bax 175C-NBD
has a larger change in NBD emission compared to Bax 3C-NBD. This
Figure 1.3 (A) NBD emission change for Bax 175C-NBD (100 nM) and Bax 3C-NBD
(100 nM) upon addition of cBid (20 nM) in the presence of liposomes (0.2 mg/mL) (B
and C) Iodide quenching data of 100 nM Bax 3C-NBD (B) or 175C-NBD (C) in solution
(gray) or in the presence of liposomes (0.2 mg/mL) and cBid (20 nM) (black).
paired with the quenching data discussed below, suggests that residue 175 of
Bax inserts into the phospholipid bilayer. As this residue is part of a larger
hydrophobic sequence believed to span the bilayer, the kinetics for this res-
idue likely represent insertion of the Bax carboxyl-terminal tail into mem-
branes. This is in accordance with data that shows residue 175C is embedded
within the mitochondrial membrane in cells (Annis et al., 2005). By using
various activators of Bax or mutations known to perturb Bax function, it is
possible to determine whether these changes affect the extent of or rate at
which Bax helix 9 inserts into phospholipid bilayers.
6. DETERMINING THE TOPOLOGY OF PROTEINS

WITHIN MEMBRANES
Fluorescence quenching by heavy atoms such as iodide can be used
to determine how exposed a fluorescently tagged residue is to the solvent.
This is due to collisional quenching that occurs when I collides with an
excited fluorophore, resulting in a loss of energy back to ground state
without emission of a photon. Typically, collisional quenching requires
direct molecular interaction with the fluorophore such that the distance
of quenching is <2 Å giving a very high resolution to detect solvent acces-
sibility (Lakowicz, 2006).
Since I quenches NBD fluorescence (Crowley et al., 1993; Lin,
Jongsma, Liao, & Johnson, 2011), this technique would be advantageous
to look at the difference of residue solvent accessibility between soluble
monomeric Bax, in the absence of activator, and membrane-bound oligo-
meric Bax, in the presence of an activator.
6.1. Iodide quenching of NBD-labeled Bax

1. As in the method for NBD emission change, the fluorimeter is set to
record NBD fluorescence, and 200 mL of 1 mg/mL liposomes are added
to 800 mL of assay buffer in a quartz cuvette. Background signal is read
with stirring until stable at 37 C.
2. NBD-labeled Bax (100 nM) and cBid (20 nM) are added to cuvettes
containing either liposomes (200 mL liposomes, 800 mL assay buffer)
for quenching of membrane bound Bax, or assay buffer only (1 mL assay
buffer), for quenching of solution Bax. NBD emission (F0) is recorded
after incubation of the sample at 37 C for 1 h.
3. Multiple quenching reactions are set up where aliquots of potassium
iodide (2 M, supplemented with 2 mM sodium thiosulfate to prevent
oxidation) and potassium chloride (2 M) stock solutions are added to

each sample so that the total ion concentration, and thus ionic strength,
in samples is the same (typically 100 mM) (Table 1.2). The NBD emis-
sion for each concentration of KI is determined (F) and collisional
quenching is calculated by the Stern–Volmer equation:
F0
¼ 1 + K sv ½Q
F
where F0 is the fluorescence intensity in the absence of quencher, F is
the fluorescence intensity at a specific quencher concentration [Q] and
Ksv is the Stern–Volmer quenching constant.
As the concentration of I increases, so does the extent of quenching as
determined by the Stern–Volmer equation, allowing the titration curve
to be fit with a line where the slope is the Stern–Volmer constant (Ksv).
The smaller the Stern–Volmer constant the more protected a residue is from
the solvent. Quenching can then be used to compare the change in exposure
of Bax residues to solvent upon the addition of an activator. Residue 3 shows
a slight decrease in protection from quenching upon the addition of cBid
(Fig. 1.3B), whereas residue 175C of Bax becomes more protected from
quenching, in agreement with this region of Bax inserting into the bilayer
(Fig. 1.3C) (Annis et al., 2005).
7. CONCLUSION
Here, four techniques have been highlighted to show how membrane
proteins can be studied by fluorescence spectroscopy. The high sensitivity of
fluorescence-based assays along with the ability to probe the dynamics of
protein:protein and protein:membrane interactions in real time lends itself
well to study a complex regulatory system such as the Bcl-2 family of pro-
teins. The methods outlined use a simplified liposome system but can be
extended to study Bcl-2 family regulation in isolated mitochondria
(Shamas-Din, Bindner, et al., 2013; Shamas-Din, Kale, et al., 2013). Addi-
tionally, interactions between proteins can be detected via FRET in live cells
using fluorescence lifetime imaging microscopy that recapitulate what we
see in vitro (Aranovich et al., 2012). Thus, fluorescence is a very powerful
technique that not only allows us to determine the core mechanism of
Bcl-2 family regulation in vitro but also allows us to extend these studies
to live cells and in vivo, bridging the gap between simplified and complex
systems (Kale et al., 2012).
Table 1.2 Iodide quenching values of NBD-labeled Bax
3Ca 175Ca
KI (mM) KCl (mM) Solution (F0/F) Membrane (F0/F) Solution (F0/F) Membrane (F0/F)
0 100 1 1 1 1
20 80 1.1351 0.0105 1.1657 0.0136 1.1158 0.0002 1.0650 0.0134
40 60 1.2645 0.0068 1.3176 0.0101 1.2140 0.0029 1.1110 0.0112
60 40 1.3812 0.0180 1.4807 0.0373 1.3397 0.0019 1.1647 0.0020
100 0 1.6224 0.0156 1.7954 0.0376 1.5294 0.0002 1.2730 0.0093
a
Values for solution and membrane quenching are plotted in Fig. 1.3C.
ACKNOWLEDGMENTS
The work in the author’s laboratory is supported by Grant FRN12517 from the Canadian
Institutes of Health Research (CIHR) to D. W. A. and B. L. J. K. is recipient of a
doctoral fellowship from the Canadian Breast Cancer Foundation, Ontario Division.
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CHAPTER TWO
Photoreactive Stapled Peptides

to Identify and Characterize BCL-2
Family Interaction Sites by Mass
Spectrometry
Susan Lee*,†, Craig R. Braun*,†,{, Gregory H. Bird*,†,
Loren D. Walensky*,†,1
*Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer
Institute, Boston, Massachusetts, USA
†
Department of Pediatrics, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA
{
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
1
Corresponding author: e-mail address: [email protected]
Contents
1. Introduction 26
2. Overview of Photoreactive Stabilized Alpha-Helices Methodology 28
3. Design and Synthesis of Photoreactive Stapled Peptides 31
4. Photoaffinity Labeling and Retrieval 34
5. Mass Spectrometry Analysis 36
6. Computational Docking 38
7. Mapping Novel Binding Sites 41
8. Conclusions and Future Directions 45
Acknowledgments 46
References 46
Abstract
Protein interactions dictate a myriad of cellular activities that maintain health or cause
disease. Dissecting these binding partnerships, and especially their sites of interaction,
fuels the discovery of signaling pathways, disease mechanisms, and next-generation
therapeutics. We previously applied all-hydrocarbon peptide stapling to chemically
restore a-helical shape to bioactive motifs that become unfolded when taken out of
context from native signaling proteins. For example, we developed stabilized alpha-
helices of BCL-2 domains (SAHBs) to dissect and target protein interactions of the
BCL-2 family, a critical network that regulates the apoptotic pathway. SAHBs are a-helical
surrogates that bind both stable and transient physiologic interactors and have
effectively uncovered novel sites of BCL-2 family protein interaction. To leverage stapled
peptides for proteomic discovery, we describe our conversion of SAHBs into pho-
toreactive agents that irreversibly capture their protein targets and facilitate rapid
Methods in Enzymology, Volume 544 # 2014 Elsevier Inc. 25

ISSN 0076-6879 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-417158-9.00002-9
26 Susan Lee et al.
identification of the peptide helix binding sites. We envision that the development of
photoreactive stapled peptides will accelerate the discovery of novel and unanticipated
protein interactions and how they impact health and disease.
1. INTRODUCTION
Alpha-helical interactions are found throughout the cell and govern
critical biological processes, such as infection (Weissenhorn, Dessen,
Harrison, Skehel, & Wiley, 1997), the immune response (Blum,
Stevens, & DeFranco, 1993), apoptosis (Sattler et al., 1997), and transcription
(Kussie et al., 1996; Fig. 2.1). The natural complexity of peptide a-helices
enables them to engage diverse cellular targets with high affinity and selectiv-
ity. Indeed, helical peptides can effectively discriminate among homologous
proteins owing to the high fidelity key-in-lock specificity afforded by their
amino acid composition. The conserved BCL-2 homology 3 (BH3) domain
of BCL-2 family proteins is a critical interaction module that mediates
A. Cell surface B. Plasma membrane
C. Cytosol and mitochondrion
D. Nucleus
Figure 2.1 The peptide a-helix is a ubiquitous secondary structural motif that mediates
a host of biomedically relevant protein interactions. Hydrocarbon-stapled peptides
modeled after bioactive helices can be generated to target and modulate protein inter-
actions from the surface to the inner nuclear core of the cell.
Photoreactive Stapled Peptides 27
member-specific communication. The canonical complex between a

proapoptotic BH3 a-helix and a surface groove on antiapoptotic members
reflects the molecular wrestling match between pro- and antiapoptotic signals
(Sattler et al., 1997). If antiapoptotic grooves are sufficient in number to bind
and sequester the proapoptotic BH3 signals, cell survival prevails. In contrast,
if the capacity to withstand proapoptotic assault is breached, cell death ensues
(Fig. 2.2). Not only did the discovery of this protein interaction paradigm pro-
vide a mechanism for apoptotic regulation, but it also informed the develop-
ment of drugs to reactivate cell death through targeted inhibition of
antiapoptotic BH3-binding pockets (Oltersdorf et al., 2005).
Given the critical roles of amphipathic alpha-helices in mediating signal
transduction, we advanced all-hydrocarbon stapling to refold bioactive
Multi-BH domain Multi-BH domain BH3-only

antiapoptotic proteins proapoptotic proteins proapoptotic proteins
Heavy cell stress Weak cell stress
BAX/BAK activation Anti-apoptotic BH3-only displacement Anti-apoptotic Anti-apoptotic BAX/BAK

by BH3-only inhibition by of sequestered BAX/BAK sequestration sequestration remain inactive
BAX BH3-only of BH3-only of BAX/BAK
BAX
BAK BAK
Oligomerization
Cytochrome c release
Cell death Cell survival

Figure 2.2 BCL-2 family proteins regulate the life and death decision of stressed cells. If
mitochondrial antiapoptotic proteins (orange) can effectively harness their C-terminal
binding pockets to trap and sequester the BH3-signaling helices of proapoptotic pro-
teins, cell survival prevails. However, with increased cellular stress, proapoptotic signals
overwhelm the antiapoptotic reserve. The BH3 domain helices of BH3-only proteins
(blue) can directly activate the essential executioner proteins BAX and BAK (gray)
and also release trapped forms from antiapoptotic inhibition by targeting the anti-
apoptotic groove.
28 Susan Lee et al.
alpha-helical peptides for use as research tools and prototype therapeutics

(Fig. 2.3A). Depending on their amino acid composition and design,
“stapled peptides” have proven to be structurally stable, protease resistant,
and cell permeable agents capable of interrogating and modulating protein
interactions in vitro and in vivo (Bernal et al., 2010; Kim et al., 2013; LaBelle
et al., 2012; Takada et al., 2012; Walensky et al., 2004). Such stapled pep-
tides have revealed unanticipated and functionally relevant helix/target
interactions, including engagement of (1) antiapoptotic MCL-1 by its
own MCL-1 BH3 helix (Stewart et al., 2010; Fig. 2.3B), (2) a novel
“trigger site” on proapoptotic BAX by the BIM BH3 helix (Gavathiotis
et al., 2008; Fig. 2.3C), and (3) glucokinase by a BH3-phosphorylated form
of the BH3-only protein BAD (Danial et al., 2008). The capacity of these
stabilized alpha-helices of BCL-2 domains (SAHBs) to access canonical and
noncanonical protein interactors, and reveal sites of interaction by their use
in NMR and X-ray crystallography studies, prompted us to consider
whether they could be harnessed for higher throughput binding site discov-
ery. Here, we describe our approach to converting stapled peptides into high
fidelity photoaffinity reagents and the methodologies developed to rapidly
identify their sites of target protein interaction.
2. OVERVIEW OF PHOTOREACTIVE STABILIZED

ALPHA-HELICES METHODOLOGY
We generate photoreactive stabilized alpha-helices or pSAHs by
substituting into the peptide sequence a nonnatural amino acid bearing
a benzophenone moiety (4-benzoylphenylalanine, Bpa) (Fig. 2.4A),
which covalently crosslinks to protein targets upon exposure to ultraviolet
(UV) light (Dorman & Prestwich, 1994; Saghatelian, Jessani, Joseph,
Humphrey, & Cravatt, 2004; Vodovozova, 2007). We then insert an
(i, i + 4) or (i, i + 7) pair of nonnatural amino acids bearing olefin tethers,
followed by ruthenium-catalyzed ring-closing metathesis (RCM), to gener-
ate a hydrocarbon-stapled peptide with reinforced a-helical structure
(Blackwell et al., 2001; Schafmeister, Po, & Verdine, 2000; Fig. 2.4B).
For binding site identification, pSAHs containing alternatively placed Bpa
moieties are individually incubated with a protein-of-interest and UV irra-
diated (Fig. 2.4C). The mixture is subjected to electrophoresis and the
crosslinked species excised, trypsinized, and prepared for mass spectrometry
analysis, which is designed to identify peptidic fragments bearing Bpa cross-
links and thus the explicit sites of target protein intercalation (Fig. 2.4D).
A
Insert nonnatural
amino acids
Cy3P
CI Ph
Ru
Chemically “staple” CI
O Cy3P
O NH
the peptide back
* OH
CH3
into shape
O
[ ]n
Unstructured peptide Stapled peptide

- Loss of functional shape - Stabilized structure
- Easily degraded - Not degraded
- Does not enter cells - Enters cells
Hydrophobic Hydrophilic
Positive charge Negative charge
B C
R214
D218
MCL-1 SAHBD
BIM BH3
K21
R263
N260
D256
S255
Staple Q32 R134
E131
MCL-1Δ NΔ C BAX
Figure 2.3 Synthetic overview of all-hydrocarbon stapling and its applications in devel-
oping a-helical peptides for structural studies of protein interactions. (A) Pairs of
a,a-disubstituted nonnatural amino acids bearing olefin tethers are substituted into
the peptide sequence at discrete locations (e.g., i, i + 4), followed by ruthenium-
catalyzed ring-closing metathesis (RCM) to generate “stapled peptides.” (B) Crystal
structure of a stapled MCL-1 BH3 helix in complex with antiapoptotic MCL-1
(Stewart, Fire, Keating, & Walensky, 2010). (C) Calculated model structure of a BIM
BH3 helix engaging the N-terminal trigger site of full-length BAX, as derived from
paramagnetic relaxation enhancement NMR analyses using full-length 15N-BAX- and
MTSL-labeled BIM SAHBs (aa 145–164) (Gavathiotis et al., 2008).
30 Susan Lee et al.
A B O Cy3P
CI Ph
O NH
* OH
Ru
CH3
CI
O Cy3P
[ ]n
O
O O
UV light Trypsinolysis
C D
O
O
E F
Figure 2.4 Design of photoreactive stabilized alpha-helices (pSAHs) for binding site
identification by mass spectrometry. A photoreactive Bpa residue (A) and a pair of sta-
pling amino acids (B) are inserted into the peptide template followed by RCM to gen-
erate a pSAH. Upon exposure to UV light, the bound pSAH covalently crosslinks to the
target protein (C) and, following electrophoresis of the mixture, crosslinked protein is
excised from the gel and subjected to in-gel digestion with trypsin (D). LC–MS/MS anal-
ysis identifies the explicit sites of covalent modification, which when mapped on to the
protein structure reveal the region of pSAH interaction (E). Top scoring crosslinks from a
series of experiments employing sequentially placed Bpa residues can provide interac-
tion restraints for calculating model structures (F).
By mapping the crosslink sites of pSAHs bearing alternatively placed Bpa

moieties onto the protein structure (if known), the location of the interac-
tion site can be determined (Fig. 2.4E). Furthermore, if the structure of the
protein target has been solved and a series of pSAHs with sequentially local-
ized Bpa residues map sites of intercalation with sufficient resolution,
computational docking can be applied to calculate a model structure of the

complex for biochemical and functional validation (Fig. 2.4F).
3. DESIGN AND SYNTHESIS OF PHOTOREACTIVE

STAPLED PEPTIDES
The design of photoreactive stapled peptides is ideally based on a
structure involving the template a-helix, so that the structurally reinforcing
staples can be placed on the noninteracting surface and Bpa residues located
at or near the putative interaction surface to facilitate UV crosslinking. Ide-
ally, Bpa residues are substituted at positions of homologous, bulky, and
hydrophobic residues (e.g., Phe) to minimize the effect of Bpa on the bind-
ing interface. Without structural information, a library of constructs can be
generated by sampling alternative staple and Bpa positions along the length
of the peptide template, selecting those constructs with successful a-helical
induction (as determined by circular dichroism analysis; Bird, Bernal,
Pitter, & Walensky, 2008) and preserved binding activity for experimental
application. Of note, to simplify interpretation of the MS2-based identifica-
tion of Bpa-crosslinked peptides, it is also preferable to locate Bpa residues in
regions of the peptide such that trypsinization will yield short cleavage prod-
ucts. If necessary, a lysine or arginine residue can be strategically substituted
into the peptide, ideally on the noninteracting surface or as a conserved sub-
stitution observed in other species (Leshchiner, Braun, Bird, & Walensky,
2013), to accomplish this goal.
Once designed, stapled peptides are synthesized as previously described in
detail (Bird et al., 2008; Bird, Crannell, & Walensky, 2011) using Fmoc chem-
istry, stapling amino acids installed at (i, i + 4) or (i, i + 7) positions (to bridge
one or two turns of the a-helix, respectively), and ruthenium-catalyzed olefin
metathesis with Grubbs generation I catalyst (Fig. 2.5). Of note, methionine
residues in the template sequence are typically replaced by norleucines, as the
unprotected sulfur can decrease the efficiency of the Grubbs catalyst. Follow-
ing peptide deprotection and cleavage, pSAHs are purified by reverse phase
high performance liquid chromatography–mass spectrometry and quantified
by amino acid analysis (AAA). Pure peptide is stored as a lyophilized powder
at 20 C until use.
In addition to the experimental conditions summarized in Fig. 2.5 and
the step-by-step synthetic protocol with reagent list detailed in our recently
updated methodologic review (Bird et al., 2011), our approach to stapled
peptide synthesis incorporates the following general principles:
32 Susan Lee et al.
R
H 1. 20% piperidine in NMP
Fmoc N
N O
H HCTU/DIEA 2. H
O
in NMP
Fmoc N OH
n
Bp
1. 20% piperidine in NMP
HCTU/DIEA O
H O
in NMP 2. N (S) R O R O R
Fmoc OH H H H H
Fmoc N N N N
N N N
H H H
Bp O O O
H
O R
H n⬘ 3 n
N N
Fmoc N
H
O
n 1. 20% piperidine in NMP
1. 20% piperidine in NMP HCTU/DIEA R

2.
in NMP Fmoc OH
HCTU/DIEA R N
2. H
in NMP Fmoc OH O
N
H
O
R O R O
H H R R O R O R
Fmoc N N H H H H
N N Fmoc N N N N
H H N N N N
O O H H H H
O Bp O O O
3 n n⬙ n⬘ 3 n
O
H
2. N (S)
Fmoc OH
HCTU/DIEA
in NMP 2. Ac2O, DIEA, NMP
O R O R
H H H O
N N N R R O R O R
Fmoc N N H H H H
H H Ac N N N N
O O N N N N
H H H H
O O O O
3 n Bp
n⬙ n⬘ 3 n
R 1. PCy3
2. Cl
HCTU/DIEA Fmoc OH , DCE
N Ru
in NMP H
O Cl
PCy3
2. TFA:TIS:H2O (95:2.5:2.5)
O
R O R O R R R O R O R
H H H H H H
Fmoc N N N Ac N N N NH 2
N N N N N N N
H H H H H H H
O O O O Bp O O O
n⬘ 3 n n⬙ n⬘ n
Figure 2.5 Synthetic steps for the automated production of pSAHs by Fmoc-based
solid-phase peptide synthesis and ruthenium-catalyzed RCM. Alternatives to
N-terminal acetylation include capping with FITC or biotin, depending on the desired
application. Bp, 4-benzoylphenyl.
a. Create an automated synthesis program by employing standard double

Fmoc deprotections, vigorous washes with DMF, and subsequent single
10-fold excess or double fivefold excess amino acid couplings. The pre-
cise timing and coupling excesses are optimized based on the specific
coupling reagents, bases, and synthesizer employed. Particular attention

is devoted to reactions involving the a,a-disubstituted amino acids, as
the amine is linked to a quaternary carbon center, and therefore, can
be recalcitrant to Fmoc removal and the subsequent acylation steps.
For example, couplings that involve beta-branched amino acids and
arginine can be especially challenging. Typical protocol modifications
include multiple extended deprotections and acylations, and sometimes
capping at these difficult steps with acetic acid (AcOH) or even decanoic
acid in order to capture and terminate unreacted amine. In this manner,
the production of full-length peptide species can be maximized, while
also avoiding the challenge of chromatographic separation of truncated
byproducts.
b. Prepare the peptide synthesis reagents and solutions by weighing out the
resin and dissolving the calculated amount of each amino acid in anhy-
drous NMP. Although more expensive than DMF, NMP has greater
stability and we have seen no deterioration in peptide quality using
reagent solutions for up to 2 weeks. A secondary amine is used to remove
the Fmoc protective group; appropriate choices include piperidine,
piperazine, or 1,8-diazabicycloundec-7-ene. Likewise, there are multi-
ple coupling reagents to select from. HCTU/DIEA and HOBt (or
HOAt)/DIC nicely balance reactivity with solution stability, and more
reactive coupling reagents range from uronium based (HATU) to the
more exotic tris-pyrrolidinophosphonium derivatives.
c. Evaluate synthetic success by subjecting a small sample of beads to stan-
dard trifluoroacetic acid (TFA) cleavage (95:2.5:2.5 TFA:water:
triisopropylsilane) and analysis by LC/MS. If the full-length species is
identified in good yield, the next step is acylation with acetic anhydride
or Fmoc-b-alanine, followed by stapling with Grubbs I catalyst (bis
(tricyclohexylphosphine) benzylidine ruthenium (IV) dichloride) dis-
solved in dichloroethane. For most sequences and staple locations, a
2 2 h incubation using several milliliters of a 4 mg/mL solution will
yield a completely metathesized product. If there is incomplete metath-
esis, the reaction can be repeated multiple times and with longer incu-
bation periods.
d. After confirmation of successful stapling, the Fmoc group of Fmoc-
b-alanine is removed and the N-terminus derivatized with biotin or
fluorescein isothiocyanate (FITC), depending on the application (see
later). The final product is cleaved off the resin in large scale, precipitated
with ether:hexanes, and purified by HPLC or, if available, by LC/MS
with mass-triggered fraction collection. The pure fractions are pooled,
34 Susan Lee et al.
lyophilized, and peptide powder resuspended in a known volume,

followed by submission of several dilutions for AAA. Although we typ-
ically store stapled peptides as dried powder (see above), the material can
also be solubilized in 100% DMSO for storage at 20 C.
4. PHOTOAFFINITY LABELING AND RETRIEVAL

With a panel of photoreactive stapled peptides in hand, the next step is
to isolate pure protein-of-interest for photoaffinity labeling. We have typ-
ically expressed glutathione S-transferase, chitin-binding domain, or
histidine-tagged fusion proteins followed by affinity purification, cleavage
of the tag, and size exclusion chromatography. Protein purity and identity
are confirmed by SDS-PAGE, protein staining (e.g., coomassie, silver),
protein-specific western blot, and mass spectrometry analyses. Protein con-
centrations are determined using the Bradford assay (Biorad).
Photoreactive stapled peptide and pure recombinant protein (e.g.,
10 mM) are typically mixed at a 1:1 ratio, but higher pSAH concentrations
can be used if necessary to increase the quantity of crosslinked product. The
peptide/protein mixture is vortexed, incubated for 20 min in the dark, and
then irradiated (365 nm) on ice for 1.5 h by use of a Spectroline handheld
UV lamp (Model En280L, Spectronics Corporation). The duration of UV
exposure can also be adjusted based on the efficiency of crosslinking for a
particular peptide/protein complex. The mixtures are then diluted with
4 LDS loading buffer (Invitrogen), electrophoresed (e.g., 4–12% Bis–Tris
gels [Invitrogen]), and analyzed by coomassie staining (SimplyBlue Safestain,
Invitrogen). The appearance of a slower migrating band just above the pro-
tein starting material reflects the pSAH-crosslinked species, which is excised
for MS analysis (see below).
Several supplementary experiments can be especially useful when
piloting this approach. For example, to confirm that insertion of staples
and Bpa residues at discrete sites within the peptide template do not disrupt
target engagement, we recommend generating fluorescently labeled (FITC)
pSAH derivatives for comparative fluorescence polarization (FP) binding
analyses with a positive control ligand. In the case of FITC-BAD pSAHBs,
the substitution of Bpa residues at four positions within the BAD BH3
sequence had no disruptive effect on binding to the antiapoptotic protein
BCL-XLDC (Braun et al., 2010; Fig. 2.6A). The FITC-derivatized pSAHs
can also be used for photoaffinity labeling, with conversion of recombinant
protein into a fluorescent species that can readily be detected by fluorescence
imaging (Gel Doc XR Molecular Imager and Quantity One software, Bio-
Rad), providing further confirmation of effective crosslinking (Braun et al.,
2010; Fig. 2.6B). Biotinylated pSAHs can also be used for high sensitivity
screening of effective crosslinking, with electrophoresed protein analyzed
by anti-biotin western blot (see below). Binding specificity experiments
using bovine serum albumin (BSA), for example, are also advised.
Crosslinking is performed as above, except that BSA is added in molar excess
Coomassie
Compound EC50 95% CI BSA
A C Crosslinked
BAD SAHB 60.5 (51.9–70.6) BCL-XLΔC
BAD pSAHB-1 46.7 (44.2–49.4) BCL-XLΔC
BAD pSAHB-2 38.9 (35.9–42.1)
Fluorescence
BAD pSAHB-3 38.5 (36.5–40.6)
BAD pSAHB-4 55.6 (53.1–58.1) BSA
Crosslinked
BCL-XLΔC
BCL-XLΔC
100
Normalized polarization (%)
BSA (mM) 10 0 .5 10 30
BCL-XLΔC (mM) 0 10 10 10 10
80 BAD pSAHB-2 (mM) 0 10 10 10 10
60 D
Coomassie
40 Crosslinked
BCL-XLΔC
BCL-XLΔC
20
Fluorescence
0 Crosslinked
10–10 10–9 10–8 10–7 10–6 10–5 BCL-XLΔC
BCL-XLΔC
[BCL-XLΔC], M
0 0.5 1 2 5 10 20 30 40 50 60 80 100 120
Time (min)
B 40
BAD pSAHB-2 crosslinking yield (%)
Coomassie Fluorescence
30
BA pS B-1
BA pS B-2
AH 3
4
BA pS B-1
BA pS B-2
Ve SA -3
BA cle B-4
pS B-
B-
p B
D AH
D AH
D AH
D AH
D AH
D AH
BA S
BA pS
BA cle
20
D
hi
D
hi
Ve
Crosslinked 10
BCL-XLΔC
BCL-XLΔC
0
0 20 40 60 80 100 120
Time (min)
Figure 2.6 pSAHs bind and selectively crosslink to their protein targets, as exemplified
by the photocrosslinking of BAD pSAHBs to antiapoptotic BCL-XLDC (Braun et al., 2010).
(A) A series of FITC-BAD pSAHBs retain nanomolar binding affinity to BCL-XLDC as mea-
sured by FP analysis. (B) FITC-BAD pSAHBs exhibit a spectrum of BCL-XLDC-crosslinking
efficiency upon UV exposure, with BAD pSAHB-2 demonstrating the greatest reactivity
toward BCL-XLDC (left, coomassie stain; right, fluorescence scan). (C) The selectivity of
BAD pSAHB-2 is reflected by the absence of crosslinking to BSA and the lack of effect of
added BSA on the BCL-XLDC-crosslinking efficiency of FITC-BAD pSAHB-2. (D) Time
course for photocrosslinking of FITC-BAD pSAHB-2 to BCL-XLDC as monitored by
coomassie stain (top) and fluorescence scan (middle). A plot of crosslinking yield, cal-
culated based on densitometry, demonstrates the time-dependent production of
crosslinked BCL-XLDC (bottom).
36 Susan Lee et al.
prior to addition of pSAH. In the case of FITC-BAD pSAHB-2/BCL-X-

LDC crosslinking, the addition of molar excess BSA had no effect on the
yield of pSAHB-crosslinked BCL-XLDC (Braun et al., 2010; Fig. 2.6C).
Tracking the production of pSAH-crosslinked protein over time is useful
for determining just how long to extend the reaction time to produce max-
imal reaction product for the MS analyses (Fig. 2.6D).
If the efficiency of pSAH crosslinking is suboptimal after increasing the
peptide/protein ratio and extending the reaction time (i.e., crosslinked species
not readily visualized by coomassie stain), the capacity to load sufficient material
for electrophoresis and band excision can be compromised. Although we typ-
ically find that sampling a variety of pSAH constructs reveals lead photoaffinity
labeling reagents (Fig. 2.6B), an alternative approach to enrich for crosslinked
species entails the synthesis and application of pSAHs capped at the N-terminus
with biotin. Photoaffinity labeling with biotinylated pSAHs is performed as
above and then unreacted peptide is removed from the irradiated samples by
overnight dialysis at 4 C in 50 mM Tris pH 7.4, 200 mM NaCl buffer using
6–8 kDa molecular weight cut-off D-Tube dialyzers (EMD Biosciences).
After addition of SDS to a final concentration of 0.2%, biotinylated protein
is isolated from unreacted protein by incubation with high capacity streptavidin
agarose (50 mL 50% slurry/reaction) for 2 h at room temperature. The
streptavidin beads are successively washed at room temperature in 1% SDS
in PBS, 1 M NaCl in PBS, and then 10% ethanol in PBS for 3 10 min each.
Biotinylated proteins are eluted by boiling for 30 min in a 10% SDS solution
(Promega) containing biotin (10 mg/mL), electrophoresed (e.g., 4–12%
Bis–Tris gels [Invitrogen]), and then subjected to coomassie staining. Using this
enrichment protocol for purifying biotinylated pSAH-crosslinked protein, iso-
lating sufficient material for MS analyses of even low efficiency crosslinked
complexes (e.g., weak or transient protein interactions) can typically be
achieved (Edwards et al., 2013; Leshchiner et al., 2013).
5. MASS SPECTROMETRY ANALYSIS

To prepare pSAH-crosslinked protein for MS analysis, we perform an
in-gel digest of the excised protein band using a standard protocol (Braun
et al., 2010), mass spectrometry grade reagents, and due caution to avoid ker-
atin contamination. Gels are subjected to coomassie or other MS-compatible
stain (e.g., Pierce Silver Stain) and the excised gel slab cut into 1 mm cubes for
processing as follows:
a. Destain: Add 500 mL of 50% acetonitrile (ACN)/50 mM ammonium

bicarbonate (AB), pH 8 and incubate at 37 C with shaking for
15 min; remove solution and repeat until the blue color is eliminated
from the gel pieces.
b. Dehydrate: Add 500 mL of ACN, incubate at room temperature for
5 min, remove solution, and repeat.
c. Reduce disulfides: Add 100 mL of 10 mM dithiothreitol in AB, incubate at
56 C for 30 min, and remove solution.
d. Alkylate cysteines: Add 100 mL of 50 mM iodoacetamide in AB, incubate
at room temperature in the dark for 20 min, and then remove solution.
e. Dehydrate: As above.
f. Digest: Cover gel pieces with cold 12.5 ng/mL trypsin (Promega) in AB,
incubate on ice for 45 min and then overnight at 37 C. Peptides are
extracted twice in 50% ACN/5% formic acid (FA) and dried in a speed-vac.
Samples are prepared for LC–MS/MS using home-made C18 stage tips or
Agilent C18 Omix tips (Braun et al., 2010; Jakobsen, Schroder, Larsen,
Lundberg, & Andersen, 2013). The sample preparation procedure below
employs 100 mL Omix tips:
a. Place 6 mL of elution buffer (80% ACN/0.5% AcOH) in a clean elution
tube for each sample.
b. Resuspend the dried peptides in 100 mL of 0.1% TFA.
c. Wet tips by pipetting 2 100 mL 40% ACN/0.5% AcOH. Be sure to
prevent air from being introduced into the tips once wetted.
d. Equilibrate tips by pipetting 2 100 mL of 0.1% TFA.
e. Bind peptides in sample by carefully pipetting up and down 10 times.
f. Wash by pipetting 3 100 mL of 0.1% TFA.
g. Elute by carefully pipetting up and down in a prepared elution tube
three times.
h. Dry the eluate by speed-vac.
We use a split flow LC system with in-house packed C4/C18 columns for
MS analysis on a Thermo LTQ Orbitrap Discovery mass spectrometer.
Samples are reconstituted in 5% ACN/5% FA and loaded onto the LC.
We employ 30 cm of 100 mm inner diameter fused silica tips packed with
<0.5 mm C4 (Sepax 5 mm, 120 Å) and then C18 slurry (Nest 3 mm,
200 Å). LC solutions are (A) 3.1% ACN/0.125% FA/water and (B)
0.125% FA/ACN. We typically employ a 2 h gradient from 10% to 40%
B with a top 10 data-dependent method.
Analysis of the MS/MS spectra can be accomplished using the
SEQUEST algorithm (Eng, McCormack, & Yates, 1994; Yates, Eng,
38 Susan Lee et al.
McCormack, & Schieltz, 1995), searching against a partially tryptic database

containing the target protein, trypsin, and common keratin contaminants.
Data analysis can also be accomplished using commercial software packages,
such as Thermo Proteome Discoverer or XTandem! Analytical parameters
include: enzyme, trypsin; missed cleavages, 2; parent tolerance, 10 ppm;
fragment ion tolerance, 0.8 Da. Tryptic peptides containing Bpa crosslinks
are identified by allowing for a variable modification corresponding to the
mass of the Bpa-containing pSAH fragment on any amino acid of each pro-
tein in the database. Of note, the reversed protein sequences are also
included in the database to generate an estimate of false-positive detection
rate. For example, the subset of crosslinked peptides identified by a
SEQUEST search are filtered based on tryptic state, charge state, mass accu-
racy, peptide length, and number of correct matches per protein, such that
the maximum false-positive detection rate is 5%. Results containing more
than one instance of photoaffinity labeling are excluded from the data set,
as the crosslinking stoichiometry is assumed to be 1:1. pSAH modifications
are further confirmed by manual examination of each MS/MS spectrum.
The resultant list of covalently modified sites is plotted by frequency of
occurrence across the polypeptide sequence of the target protein. Fortu-
nately, we have found that, if anything, the relative inefficiency of photo-
affinity labeling with pSAHs results in the identification of too few rather
than too many covalently modified sites, yielding reproducible and highly
specific results—especially for relatively stable protein interactions. For
example, our earliest crosslinking analysis of a photoreactive BAD pSAHB
with antiapoptotic BCL-XLDC, identified a focal cluster of covalently mod-
ified sites with F105 emerging as the most frequently crosslinked residue
(Fig. 2.7A). To depict the crosslinking “hot spots” on the protein structure,
a color scale is assigned by converting crosslinking frequencies to percent of
maximum occurrence and then colored by groups of 10 percentiles. When
we compared the previously reported crystal structure of the BAD BH3/
BCL-XLDC complex (PDB ID 2BZW) with our crosslinking results,
F105 indeed represented the nearest neighbor to Y110 of the BAD BH3
helix, the exact site of our Bpa substitution (Fig. 2.7B).
6. COMPUTATIONAL DOCKING
When the structure of a target protein-of-interest is known but the site
of a putative peptide helix interaction site is not, our pSAH photoaffinity
labeling/MS approach can be combined with computational docking to
A
BAD pSAHB-2
Biotin–βAla–NLWAAQRUGRELRXBSDXFVDSFKK
35
30
Crosslink occurrence
F105 F105
25
20
15
10
5
0
1
16
31
46
61
76
91
6
1
6
1
6
1
6
1
6
10
12
13
15
16
18
19
21
22
BCL-XL ΔC
B
BCL-XLΔC
Phe105
Tyr110
BAD BH3
Figure 2.7 pSAHs localize helix/target interaction sites with high fidelity, as exemplified
by the capacity of BAD pSAHB-2 to accurately map the BAD BH3 interaction site on BCL-
XLDC (Braun et al., 2010). (A) The plot (left) depicts the frequency of crosslinked sites
identified across the BCL-XLDC polypeptide sequence. Mapping of the crosslinked res-
idues onto the BCL-XLDC structure (right) revealed their striking colocalization within a
circumscribed region of the canonical BH3-binding pocket, with the frequency of occur-
rence reflected by the color scale. Green arrowheads, trypsin digestion sites. (B) The
most abundant crosslink, located between BAD pSAHB-2 Bpa110 and BCL-XLDC
F105, precisely matches the structurally defined interaction between BAD BH3 Y110
(cyan) and BCL-XLDC F105 (yellow) at the BH3-binding pocket of BCL-XLDC (PDB ID
2BZW). U, Bpa; X, stapling amino acid; B, norleucine.
calculate model structures of helix/target complexes for biochemical, struc-

tural, and functional validation. This approach is also useful for validating
new pSAH reagents on established targets before exploring their putative
binding interactions with novel targets. For example, to validate the binding
specificity and utility of a BID pSAHB panel, we conducted crosslinking
analyses with BCL-XLDC, employing pSAHBs with sequentially placed
benzophenone moieties. Data analysis for each BID pSAHB construct
40 Susan Lee et al.
revealed high regiospecificity of modification sites, enabling the calculation

of a model structure (Fig. 2.8).
Computational docking analyses are performed using crystallography
and NMR system solve (CNS) (Brunger et al., 1998) within HADDOCK
2.0 (Dominguez, Boelens, & Bonvin, 2003). Starting structures are based on
reported structures of the helical ligand and protein target of interest, or their
BID pSAHB-1
Biotin–βAla–EIIHNIXRHLXQIGDEBDRNUQ
10
Crosslink occurence
8 I101Bpa
6
4 BID BH3
2
0
0
0
0
0
0
0
0
0
0
0
0
10
20
30
40
50
60
70
80
90
10
12
13
14
15
16
17
18
19
20
11
BCL-XL residue number
BID pSAHB-2
Biotin–βAla–EIIRNUARHLAXIGDXBDHNIQ
8
Crosslink occurence
0
110
130
140
150
160
170
180
190
200
0
120
10
20
30
40
50
60
70
80
90
10
BID pSAHB-3
Biotin–βAla–EIURNIXRHLXQIGDEBDHNIQ I83Bpa
2
Crosslink occurence
I86Bpa
1.5
1
BCL-XLΔC
0.5
0
0
0
0
0
0
0
0
0
0
0
0
10
20
30
40
50
60
70
80
90
10
12
13
14
15
16
17
18
19
20
11
Figure 2.8 The high regiospecificity of pSAH crosslinking enables the calculation of mod-
el structures of helix/target complexes, as exemplified by a panel of BID pSAHBs cross-
linked to BCL-XLDC (Leshchiner et al., 2013). The plots (left) and the corresponding
mapping (right) reflect the frequency of crosslinked sites across the BCL-XLDC poly-
peptide sequence for three distinct BID pSAHBs. The covalently modified residues for
each pSAHB construct maps to a highly circumscribed subregion of the canonical
BH3-binding pocket of BCL-XLDC, facilitating the calculation of a model structure
of the complex (right) using CNS within HADDOCK 2.0. Orange arrowheads, tryptic
digestion sites; U, Bpa; X, stapling amino acid; B, norleucine.
close homologues. Models are generated by docking calculations that

employ interaction restraints based on the top scoring crosslinks. For exam-
ple, force constants for the restraints are set to 10 kcal mol 1 Å 2 for the
rigid-body docking stage and then set to a final value of 50 kcal mol 1 Å 2
during the semiflexible simulated annealing stage. The restraints are set to be
satisfied
P 6 as1/6 soon as any one of the distances entering the sum average
([ 1/r ] ), over all individual pairwise combinations, is within the
defined cut-off distance of 2 Å. The docking calculations are performed
with standard HADDOCK protocols (Arnesano, Banci, Bertini, &
Bonvin, 2004). Initially, 2000 orientations/structures of the complex are
generated by rigid-body docking energy minimization of the individual
structures. At this stage, structures are docked based on the restraints, van
der Waals, and electrostatic energy terms. The 100 better-scored structures
are semiflexibly refined in torsion angle space and then refined in explicit
water (Linge, Habeck, Rieping, & Nilges, 2003). Final structures are then
calculated following water refinement and energy minimization. During the
simulated annealing and water refinement, all amino acids (side chains and
backbone) of the peptide helix are allowed to move to optimize the interface
packing. In addition, at this stage, specific segments of the target protein
structure that comprise the binding pocket are fully flexible (side chains
and backbone). The 20 best-scored structures from the water refinement
step are clustered into one group using pairwise backbone root mean square
deviation (r.m.s.d.) of 3 Å. The final structure ensemble is evaluated for chi-
rality and stereochemistry with the programs WHATCHECK (Hooft,
Vriend, Sander, & Abola, 1996) and PROCHECK_NMR (Laskowski,
Rullmannn, MacArthur, Kaptein, & Thornton, 1996), and then displayed
and analyzed using PYMOL (DeLano, 2002).
7. MAPPING NOVEL BINDING SITES

In recent years, we have deployed the pSAH technology to identify
novel BCL-2 family interaction sites, informing both canonical and non-
canonical mechanisms of the cell death pathway. The complexes between
a series of proapoptotic BH3 helices and a binding pocket at the
C-terminal face of antiapoptotic proteins were the first BCL-2 family inter-
actions to be structurally and functionally defined (Petros, Olejniczak, &
Fesik, 2004). This work demonstrated how antiapoptotic proteins can
42 Susan Lee et al.
intercept and neutralize proapoptotic BH3 signals and vice versa (Fig. 2.2).
The question of how the essential executioner proteins, BAX and BAK, are
triggered, however, remained an enigma. One theory was that proapoptotic
BH3 helices could directly engage BAX and BAK to activate their pore-
forming function (Wei et al., 2000; Fig. 2.2). To probe this hypothesis,
we subsequently developed stapled peptides modeled after the BID and
BIM BH3 helices (Walensky et al., 2006), identified a new binding site at
the N-terminal face of full-length BAX by NMR spectroscopy
(Gavathiotis et al., 2008), and correlated engagement of this site to structural
and functional activation of BAX in vitro and in cells (Gavathiotis, Reyna,
Davis, Bird, & Walensky, 2010; Gavathiotis et al., 2008).
Stimulated by these findings, we sought an approach that harnessed sta-
pled peptides to rapidly and accurately map binding interfaces. Our goal in
developing the pSAH technology was to bridge the gap between discover-
ing protein interactions and defining biologically relevant interfaces by
structural methods, which often takes years to accomplish. With pSAHs
in hand, we turned our attention to BAK, a BAX homologue that had been
refractory to protein expression and structural evaluation in full-length form,
likely owing to its constitutive membrane disposition in the cell. After over-
coming the challenge of producing pure, monomeric, and functionally
active full-length BAK protein (Leshchiner et al., 2013), we applied BID
pSAHBs and explicitly mapped the BID BH3-activating interaction to a
binding pocket at the C-terminal face of the BAK protein, mirroring the
location of BH3 interaction sites on antiapoptotic targets (Fig. 2.9A). The
subsequent NMR analysis of a stapled BID BH3 peptide in complex with
a BAK construct lacking its C-terminal helix (BAKDC) provided a defini-
tive structure that corroborated the BID pSAHB results (Moldoveanu et al.,
2013). Interestingly, the interaction site analysis of full-length BAK protein
did not detect BH3-engagement at the N-terminal face of BAK, yet the
same BID pSAHBs peppered the N-terminal face of full-length BAX with
covalent modification sites corresponding to the N-terminal “trigger site”
that we previously identified by NMR analysis (Gavathiotis et al., 2008;
Leshchiner et al., 2013). These data suggested that the N-terminal trigger
site on BAX may reflect a unique and afferent direct activation mechanism
required to translocate BAX from the cytosol to the mitochondria, a process
not required for BAK given its constitutive localization at the mitochondrial
outer membrane.
Further interrogation of this hypothesis using pSAHs revealed that both
BID and PUMA BH3-based constructs predominantly crosslinked to the
A
BID pSAHB-1 BAK: C-terminal view BAK: N-terminal view
Biotin–βAla–EIIHNIXRHLXQIGDEBDRNUQ
50
40
30
20
10
0
110
120
130
140
150
160
170
180
190
200
0
10
20
30
40
50
60
70
80
90
10
BAK residue number

BID pSAHB-2 BAK: C-terminal view BAK: N-terminal view
Biotin–βAla–EIIRNUARHLAXIGDXBDHNIQ
50
40
30
20
10
0
110
120
130
140
150
160
170
180
190
200
0
10
20
30
40
50
60
70
80
90
10
BAK residue number

B
PUMA pSAHB-2
Biotin–βAla–QUAREIGAQLRXBADXLNAQYE BAX: N-terminal view BAX: C-terminal view
10
0
10
20
30
40
50
60
70
80
90
110
0
0
0
0
0
0
0
0
0
10
12
13
14
15
16
17
18
19
BAX residue number
PUMA pSAHB-2
Biotin–βAla–QUAREIGAQLRXBADXLNAQYE BAXΔC: N-terminal view BAXΔC: C-terminal view
40
30
20
10
0
10
20
30
40
50
60
70
80
90
0
0
0
0
0
0
0
10
11
12
13
14
15
16
BAXΔC residue number
Figure 2.9 pSAHs identify novel sites of BCL-2 family protein interaction. BID,
PUMA, and phospho-BAD pSAHBs revealed BH3 interaction sites on (A) full-length BAK
(Continued)
44 Susan Lee et al.
C
BAD pSAHBA (Y110Bpa S118pS) GK: front view GK: back view
NLWAAQRUGRELRXBpSDXFVDSFKK
100
80
60
40
20
0
0
0
75
0
15
22
30
37
GK residue number 45
BAD pSAHBA (S118pS D123R F125Bpa)

GK: front view GK: back view
NLWAAQRYGRELRXpSDXFVRSUKK
100
80
60
40
20
0
0
75
0
15
22
30
37
45
GK residue number
Figure 2.9—Cont'd (Leshchiner et al., 2013), (B) full-length versus C-terminally trun-
cated BAX (i.e., BAX vs. BAXDC) (Edwards et al., 2013), and (C) glucokinase (Szlyk
et al., 2014), respectively. U, Bpa; X, stapling amino acid; B, norleucine; pS, pho-
sphoserine; orange R, single Arg substitution to facilitate tryptic digestion of pSAHBs
into shorter and more identifiable fragments by MS.
N-terminal BH3 interaction site on full-length BAX, with occasional cova-

lent modification sites also detected at the C-terminal binding pocket
(Edwards et al., 2013; Leshchiner et al., 2013; Fig. 2.9B). The corresponding
NMR analyses consistently showed chemical shift changes at the N-terminal
activation site upon BH3 ligand engagement (Edwards et al., 2013;
Gavathiotis et al., 2008), with C-terminal helix changes becoming apparent
at later time points and with increased ligand concentrations (Edwards et al.,
2013; Gavathiotis et al., 2010). Thus, we hypothesized that allosteric expo-
sure of the C-terminal pocket as a result of N-terminal site triggering reveals
a secondary BH3-binding site, which may function to propel BAX activa-
tion at the mitochondrial membrane in a manner analogous to BAK. Indeed,
we find that if the C-terminal helix of BAX is removed (i.e., BAXDC),
bypassing the N-terminal triggering step and providing ready exposure to
the C-terminal binding pocket, the balance of PUMA pSAHB crosslinks
Another random document with
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"I'm afraid you've been very bad sir, lately," she said.
"There's a good many of us have seen it. You've lost a deal
of flesh."
Hardly any answer came to this. The water was boiling

at last—evidenced by the straight rush of steam from the
spout and by the dancing lid. A few minutes more, and Mr.
Wilmot was gratefully sipping a cup of excellent tea.
"You certainly are an adept in the art of tea making," he

said.
"And I'm sure I'm proud to make it for you; sir,"

asserted Mrs. Stuart, with a geniality of manner which
would have astonished many of those acquaintances who
knew her only as a human icicle. An icicle needs to be
thawed before it can possibly become warm; and not many
people in Littleburgh possessed such thawing powers as Mr.
Wilmot.
"Sit down, Mrs. Stuart. Pray don't stand," he said kindly.
Mrs. Stuart complied.
"And you'll eat something—won't you now?" she

entreated. "My bread's every bit home-made, and I'll
answer for it as it's wholesome. You do look better for the
tea, sir, already. I didn't like to see you as you was when
you come in."
"Mrs. Stuart, if you see my daughter, don't mention this

to her," said Mr. Wilmot. "She is anxious enough already."
"No, sir. She'd need be anxious, I'm sure," said Mrs.

Stuart, unaware that it is often by no means the height of
wisdom to tell an invalid how ill he is looking. "For I don't
know as I ever did see anybody change for the worse, sir, in
a few months, as you've done—and you as used to be so
strong! Why, you've grown as thin! And not a bit of strength
in you."
"Not very much sometimes," admitted Mr. Wilmot. Then

with a grave smile he added, "But isn't it a happy thing to
be able to say, not only 'To live is Christ!' but also 'To die is
gain!'"
"Mercy, sir! You're not a-going to die," exclaimed Mrs.

Stuart, though she had often of late asserted a belief in the
fatal nature of Mr. Wilmot's illness.
"That neither you nor I can tell. It will be as my Master

wills. If He calls me, I am ready."
"But, sir—"
Mrs. Stuart stopped. Something in his look affected her

strangely. She might talk to others in a glib style about his
failing health, assuming to possess a gift of foresight, yet all
the while not fully believing her own words. To hear him
speak thus was another matter. A lump in her throat
checked utterance.
"It matters little—if one is ready—whether the call

Home comes a few years earlier or later," mused Mr.
Wilmot. "But—if one were not ready—"
"I'm sure," said Mrs. Stuart huskily—"I'm sure it wasn't

that as I meant, though I did say to Mrs. Mason as I'd never
seen nobody so changed, and Mrs. Mason said—she says—"
Actually a tear rolled down Mrs. Stuart's cheek, and fell

on her lap. Ashamed, she turned away her head.
"My kind old friend!" Mr. Wilmot said quickly, touched by
the sight. "But I did not mean to distress you, Mrs. Stuart. I
was speaking then in general terms—about you or me or
anybody; not about myself alone. The call may come to any
one among us, any day; and I should like to feel that you
and all are indeed ready for it."
Then passing naturally to another subject, he asked,

"How is Archie?"
"He's well, sir," said Mrs. Stuart, heaving a deep sigh.
"Getting on at the works?"
"Yes, sir."
"And a good son to you, Mrs. Stuart?"
"Yes, sir." Mrs. Stuart's tone grew more dubious, and

also harder. "I don't complain."
"Have you anything to complain of?"
"He's got his faults," said Mrs. Stuart stiffly.
"Why, yes—he would hardly be human if he had not,"

said Mr. Wilmot, smiling. "What of his liking for bright little
Nancy Dunn?"
Mrs. Stuart's face became grim. All softness and

geniality had died out of it.
"She is a good girl, Mrs. Stuart," said the Rector.
"She ain't the sort for me!" said Mrs. Stuart.
"I am sorry for that. She seems to me just the sort for
your Archie."
Mrs. Stuart was silent.
"Another person can, perhaps, hardly judge for a

mother in such a matter," observed Mr. Wilmot. "But take
care, Mrs. Stuart. It is a serious responsibility for you to
refuse your consent, if there is not a sufficient reason. I
have wished for some time to have a few words with you on
this subject."
"Yes, sir," said Mrs. Stuart, with unsmiling visage.
"Don't count it interference on my part. You and I are

old friends, and I am thinking about your son's happiness.
Archie is, I do believe, earnestly seeking now to serve God;
and Nancy Dunn is one who would help him on in the right
path. If you stop the thing altogether, Archie's next fancy
may be for a very different kind of girl. I want you to think
over the idea. Archie is no longer a child at your knee, and
sooner or later he must decide for himself. I hope he will
not go against your will; but it is very important that your
will should be distinctly on the right side. Probably years
may pass before Archie will be in a condition to marry.
Meantime, I can scarcely fancy any greater help in keeping
him steady, than that he should be engaged to such a girl
as Nancy."
Mrs. Stuart was silent.
"Come, you will reconsider the matter, perhaps," said

Mr. Wilmot, standing up. "Second thoughts are often best.
Thank you very much for your nice tea. I must not wait
longer, for somebody has appointed to see me at home.
Good-bye, Mrs. Stuart."
"Good-bye, sir," said Mrs. Stuart.

Mr. Wilmot turned back on the threshold to say in a kind
tone—"I wish you would give your consent."
Then he was gone.
CHAPTER XXV.
REST.
"FATHER, I expected you home nearly an hour ago,"

said Annie, meeting Mr. Wilmot at the hall door.
"Yes—I could not come, dear."
"Mr. Page has been waiting ever so long in the study to

see you."
There was an almost imperceptible sigh. "I should have

been in time, but I was stopped. One of the poor little
Handcocks has been terribly scalded. Of course I went at
once to see."
"Oh, how dreadful! How was it?" asked Annie. She was
watching anxiously her father's face, even while keeping up
in resolute style the cheerful manner which she had
cultivated of late.
"The mother out, of course, and the four infants locked

indoors alone. A kettle of water had been left on or close to
the kitchen fire, and the eldest child pulled it over, trying to
lift it farther away. The youngest child, crawling on the floor,
narrowly escaped a complete deluge. Bad enough as it is."
"Who helped the poor things?"
"Nancy Dunn. Her mother was out, and Nancy behaved

admirably. Bess Gardiner went off in search of Mrs. Mason."
"Bess Gardiner! Why, how was she not in the factory?"
"Some repairs going on in the machinery where she

works. Annie, dear, I think I must sit down."
"O father!" Annie's start was self-reproachful. "How

wrong of me to keep you here! Come to the drawing-room.
Mr. Page is in the study."
"Better get that over. He has waited so long.
"I wish you would not. I am sure you ought to rest."
Mr. Wilmot stooped to kiss Annie.
"Nothing of consequence, I think," he said, as if

answering her unspoken fear. "We will have a quiet evening.
Come to me when Mr. Page goes."
But Mr. Page was sure to be long in going. Annie knew

this well from past experience. She saw her father
disappear within the study door, dragging one foot after the
other. Then she busied herself in the drawing-room as best
she could, waiting for the welcome sound of footsteps in the
hall.
How Mr. Page's voice went on—on—on! Annie could

seldom hear the sound of her father's voice in response. Mr.
Page seemed to have a large amount to say; and he was
very lengthy in the saying of it.
Suddenly a ring at the front door, and the opening of

the study door. Mr. Page appeared alone, not followed as
was usual by Mr. Wilmot.
"Good evening, Miss Wilmot—good evening," said Mr.

Page. "Fine day, isn't it? Mr. Wilmot doesn't seem quite the
thing, though—no, certainly not quite the thing. He'll be
getting away for a holiday soon, and that'll set him up. I tell
him, he wants a holiday, for he works too hard—a great
deal too hard. Never any rest, morning, noon, or night,
Sunday or week-day. Human nature wasn't made to stand
it. I'm sorry to have had to stay so long, but there was a lot
of things to settle. Good-bye, good-bye."
Mr. Page vanished, and a voice at the front door was

requesting to "see Mr. Wilmot." Annie waited to see who it
might be, then glided into the study.
The room was getting rather dark, and Mr. Wilmot had
chosen a shady position, leaning back in his easy-chair.
"Father, Mr. Page has been very long. Somebody else

wants a word with you now."
A pause, and then—"I think—I can do no more."
The voice was not entirely natural.
Annie bent over him, trying to see his face.
"Are you very tired, father?"
"Yes—strangely weary to-night."

He did not ask who had come. Annie had never before
known such an omission.
"I will settle it," she said, and she went to the maid,
waiting outside the study door.
"The man must come again to-morrow," she said. "My

father is not well, and he must be quiet this evening. And
please go yourself at once for Mr. Rawdon. If he is not in,
leave a message, asking him to come as soon as he
possibly can."
Once more in the study, Annie sat down close beside

her father, watching him steadily in the dim light. He did not
appear to notice her, until she laid her hand on his, and
then his fingers closed round it.
"Father, have you any pain?" she asked.
"No, my child. Nothing, except weariness."
"When did that come on?"
He hesitated, as if to recall—then said only—"I don't

know."
His head was drooping, as if he had not strength to hold

it up. Annie put her arms round him supportingly.
"Would you not like to lie down?"
No reply came.
"If you could get a little rest—"
"This is rest," he said dreamily.

Some minutes went slowly by. It was impossible that
Mr. Rawdon should arrive yet. Annie did not like the heavy
silence. It filled her with a nameless dread.
"Father, I think you want food. May I get something?"
"No, darling. I am very comfortable."
"Really comfortable? You don't feel ill? Is it only rest

that you want?"
"Only—rest," he said.
Another prolonged silence.
"The depths of His mercy—" she heard at length

whispered.
"Yes, father—"
But silence again.
Was that the front door bell? Had Mr. Rawdon come?
She could not move to ring or ask. Mr. Wilmot was

leaning against her, supported by her in a measure. It was
as much as she could do to hold him up.
Steps sounded in the hall, and at the same instant Mr.

Wilmot stirred. Annie was conscious of a slight start—was it
from pain? She could not tell. Two words fell from his lips
with a singular distinctness, an intonation of surprise and
joy—
"READY—MASTER—"
Mr. Rawdon entered the study. He walked straight
towards the arm chair, took Mr. Wilmot's hand, dropped it,
turned to the table, and struck a light.
Annie noted first the changed look on Mr. Rawdon's

face. Then, as he relieved her of the weight she bore, she
saw her father's face.
No signs of pain there, or even of weariness now. The

eyes were lifted, looking upward, far beyond the enclosing
walls of that small room, and the pale lips, smiling, said
once more with exultant clearness—
"AYE—READY!"
Then he seemed to fall quietly asleep. It was a sleep

from which no earthly power, no human skill might awake
him.
CHAPTER XXVI.
A LOSS.
ALL Littleburgh mourned for Mr. Wilmot, for he had

been a friend to all.
Of Annie's grief it is needless to speak. The blow almost

crushed her. She felt herself alone in the world, bereft of
him who had been father, mother, friend, and companion to
her. She had indeed other friends, and a choice of homes
for the future, but none could ever fill his place in her heart.
Yet, in the worst of her woe, Annie could not but be

thankful. She could not but know how very tenderly her
Heavenly Father had dealt with them both. When she
thought of the terrible death which might have been his
portion, this most childlike falling "asleep in the Arms of
Jesus" did indeed seem only mercy.
Almost all Littleburgh went to the funeral. So great a

throng had never before been seen in the cemetery. Few
dry eyes could be perceived throughout the concourse of
people, and more than once a widespread burst of weeping
drowned the voice of the clergyman who read the service.
For Arthur Wilmot had given up his life for others; had
spent lavishly time and powers, money and strength, upon
those who needed help. No marvel that years of outpoured
sympathies should have brought a wealth of love in return.
Mrs. Stuart like others had been to the cemetery, and

like others she had wept there freely for the friend whose
face she could never see again on earth. Since returning
home, she had sat long in thought, giving vent to no
remarks.
Archie had noted his mother's gravity and absence of

mind since the day of Mr. Wilmot's death—not grimness,
which was usual, but a softened gravity which was unusual.
He noted too that her manner of speaking was gentler,
more humble, less stiff and self-asserting. He felt sure that
she was grieving deeply over the loss they had all
sustained.
"It was a wonderful sight this afternoon, mother, wasn't
it?" he said, to break in upon a silence which lasted long.
Archie had been to the funeral as well as his mother: and so
had many scores of working-men, set free from work for
the purpose by their employers.
"Ay," she said, with a deep sigh.
"There wasn't a soul stayed away that could manage to

go," pursued Archie.
"Ay," repeated Mrs. Stuart. "'Twould have been a deal

more wonderful if they hadn't gone, after all he's been and
done."
"I'm afraid we'll never get another Rector like him,

mother."
Mrs. Stuart sighed again.
"And to think of his being bitten after all by that dog,—

and nobody knowing all this while what might come of it,"
said Archie; for the fact had become known in the town.
There was no longer any reason for concealing what had
occurred. It was well that the people should know to the full
the brave and self-sacrificing spirit of the man who so long
had lived among them. If any more were needed to make
them cling to the memory of their beloved Rector, that
"more" was now supplied.
He had not, indeed, in one sense, died from the results

of the bite or scratch; but in another sense, he perhaps
had, since without it, the break-down of his health might
have been long postponed. There could be no question as to
the tremendous and terrible peril which he had willingly
incurred for the sake of helpless little ones.
"It isn't many would do what he did," said Mrs. Stuart.
"It isn't many would have thought of such a thing,

either," added Archie. "I'm sure I shouldn't. But I liked that
what the preacher said to-day, mother, about Mr. Wilmot
being willing to give up his life for the children, and about
God's pity being greater, and the Son of God giving up His
life for us. It seemed to bring the thought out so clear. I
don't know as I ever saw it so plain before. And I can
remember Mr. Wilmot telling us how pretty near all the
kindness and pity we do see round us was learnt from
Christ."
Mrs. Stuart said "Yes" thoughtfully. After a pause, she

added, "You put your life in danger too, Archie, for the sake
of Nancy Dunn."
Archie was very much astonished at such a remark from

his mother. He did not, however, show his astonishment,
but said only—"It wasn't for Nancy, mother. I didn't see her
to be Nancy till after. I hope I'd do all I could for any girl in
danger—though what I did wasn't what Mr. Wilmot did."
"But you'd sooner help Nancy than any other girl?" said
Mrs. Stuart.
"Yes—I would," said Archie, his face kindling. "Mother, I

love Nannie so much—I do think I could die for her."
"That's easy enough said," rejoined Mrs. Stuart. "It

wouldn't be so easy if you'd got to do it. But you're a brave
lad, I know. And as the preacher told us to-day—it's one
thing to be willing to die for one that loves us, and another
thing to die for them that hates us. There wouldn't be many
who'd do the last."
"Only Christ," said Archie, wondering anew at his
mother's mood.
"Only—Christ," she repeated. "Yes—He did. I don't know

as I've hated Him, like some do—but I haven't cared. I
haven't thought about Him; and that's bad enough, after all
He's done. And I hope I shan't go on so—not any longer.
You needn't tell what I'm saying—not to anybody, Archie.
But it did seem as if I must say it to somebody. And Mr.
Wilmot 'll never come again. He'd have helped me—and
there's nobody now."
"Mother, there's One," said Archie shyly; "there's One

who'll help, if anybody asks Him—and He's best of all. Mr.
Wilmot would tell you to go to Him."
"But if He shouldn't want me?" she said.
"I shouldn't think there's any chance of that, mother,"

said Archie. "I should think, if He cared enough about you
to die for you, when you weren't even born—why, it isn't
likely He'd stop caring, and turn away, just the minute
you're beginning to want to know Him."
"No—I think you're about right," said Mrs. Stuart. And

she stood up slowly, left him alone, and went upstairs to her
bedroom for a while. There are times when one needs to be
all alone with God.
CHAPTER XXVII.
HOW THINGS CAME ABOUT.
THE next evening Mrs. Stuart asked abruptly of her son
—
"Seen any of the Dunns to-day?"
"Just for a minute." Archie questioned with himself what

might be coming next.
"Nancy Dunn?"
"Yes, mother. I'd hardly a word with her, though."
"And you're hankering after her just as much as ever?"
"Mother, there isn't another girl like Nancy in all the

whole world! I give you my word for it," said Archie
earnestly.
Mrs. Stuart began to sob.
"It isn't my way to give in," she said. "I'm not one o'
those folks that's for ever chopping and changing. When I
says a thing, I mean it, and I keep to it. And I did say I'd
never have you marry Nancy Dunn. I didn't think her good
enough. Nor I don't. Leastways, her father isn't your
father's equal."
"Oh, bother!" broke out Archie.
"Yes, it's easy to say 'bother!'" she retorted, rather

disposed to flare up. She had been somewhat irritable all
day—no unusual state of things in the earlier stages of
trouble, or of any marked change in heart and life. "But if
you'd have a bit of patience and hear me out, you'd maybe
speak in a different sort of way."
"Yes, mother," Archie said meekly, a sudden hope
springing up.
"I did say I wouldn't have it," repeated Mrs. Stuart;

"and I meant it too, and I'd have kept to my word. But he—
he came in to see me, just the very same day he died—he
did, Archie, that very afternoon. And I made him a cup o'
tea, and he said—he said—I was the best tea-maker—"
"Yes, yes, mother, I know," said Archie, as she broke

out crying. "You told me all that."
"And if I did," she retorted, "there was something else I

didn't tell you nor nobody, and didn't mean to neither, till I'd
made up my mind what to do. And I couldn't make it up till
yesterday, when I was standing in the crowd, and all of 'em
in black, and everybody crying round me, and Nancy Dunn
with her pretty face all blistered—for there's no denying
she's got a pretty face."
"Yes," assented Archie, with much warmth.
"It gave me a sort of a fellow-feeling with her, and I

won't deny it," said Mrs. Stuart. "And it sort of made me
think of his talking to me like that, about you and she, and
how he hoped she'd be a good wife to you one day."
"Did he?" cried Archie.
"Yes, he did," said Mrs. Stuart, heaving another sigh.

"And I won't deny as I was a bit grumpy, not thinking as it
was the last time I'd ever see him again. O dear, dear me!
To be sure! And the very last words he says, as he was
going out of the door—looking so bad, for all he was better
for the tea, as I'm sure I'd a foreboding in my mind it
couldn't be long—and the very last words he says to me
was how he wished I'd give in and let it be."
"And you will, won't you?" begged Archie.
"I'm not one to give in easy," repeated Mrs. Stuart. "But

seeing it was, as one may say, his last wish, it do make a
difference. And I won't deny neither that I've maybe been
too stiff; and got to learn to be different. And understanding
you don't mean to think of marrying for many a year to
come—"
"Marrying! No," cried Archie. "Not yet, mother. Not till

I'm earning enough to keep you and she too in comfort. But
I do want to have her promise. I want to know she's safe to
be my wife by-and-by."
"Folks chop and change," said Mrs. Stuart. "I'd sooner

you'd keep yourself free. Maybe you'll get tired of it, or
she'll change her mind. But there, you're set on the girl,
and he wanted me to give in, and I'm not going to stand
out against you no longer. So you just go and see Nancy
Dunn, and say whatever you like, and have it settled."
Perhaps the consent might have been more graciously

worded; but Archie was far too glad to be critical. He felt
almost ashamed of his own gladness, at a time when
everybody else was sad; yet doubtless it was only natural,
and it did not prevent his sharing in the sorrow of others for
the loss they had all endured.
That same evening, Archie made his way to Woodbine

Cottage, and told the Dunns about his mother's newly-given
permission. Nannie looked hardly so pretty as usual, for her
face was still blistered with many tears, and they
threatened to flow fast again when she heard of Mr.
Wilmot's last call on Mrs. Stuart.
"It don't seem as if we'd ought to think about ourselves

yet," she faltered. But Archie could not take that view of the
question.
"I've waited ever so long, and mother's willing at last,"

he said. No doubt the few months that he had been
acquainted with Nancy did really seem long at his age. "You
won't put me off, will you, Nancy? Say she won't, Mrs.
Dunn. I do want to have it a settled thing. And you've as
good as told me already you'd say 'Yes' if my mother didn't
make difficulties. I don't mean that I'm thinking of marrying
yet awhile. I've got my mother to keep, you know. But by-
and-by—"
"It'll have to be a good long by-and-by, I shouldn't

wonder," observed Dunn. "Mind you, Archie, I'm willing to
have it all fair and straight between you and Nannie. I do
believe it'll be for her happiness and yours too, please God.
There was a time when I wouldn't have been so willing. But
I do believe it's your wish now to be a servant of God."
"Yes," Archie answered heartily. "And if I may have

Nannie, why, I hope she and I'll be able to serve God
together, Dunn."
"That's it, lad. If I didn't think so, I'd be loth enough to

give her to you. But look you here, there's something else
I've got to say. You've got your mother to keep, and it's
right you should do it. She cared for you, and you must
care for her."
"But I hope I'll soon be making enough to keep her and

a wife too," said Archie.
"Maybe," responded Dunn. "You're a capable young

fellow, and you're steady, and I hope you'll do well. Seems
no reason why you shouldn't. Only mind this, Archie, you
don't marry our Nannie, till you've got a right good sum laid
by in the Savings Bank against a rainy day. It is all very fine
to be making enough to live on in comfort, and to spend
every penny of what you've got, never giving a thought to
the future. And if illness comes, or an accident lays you by,
or trade grows slack and work fails, what's to be done then?
No, no, I'll never give in to that for Nannie. I've seen
enough of the misery of it for wives and children, let alone a
man himself. If you're bent on marrying her, you may do it;
but you'll marry with a good provision laid by; and, what's
more, you'll not squander it all away on a fine wedding, nor
on a lot of smart furniture."
Archie was well content to bind himself by these

conditions. Perhaps, like many young men, he thought them
just a little needless. In his young vigour, he could not yet
believe that bodily strength would ever fail him. But after
all, he knew that Dunn was right, and he knew that his
mother would put no difficulties in the way. Before winter,
he would be receiving the wages of a full-blown artisan, and
then, as she had often told him, would be the time to lay
by. Archie had not quite seen the necessity till now.
Laying by for the future must of course mean some

measure of self-denial. It meant this often in Archie's case.
What if it did? His love for Nannie would have been a very
poor sort of article, If it could not have endured the least
touch of self-denial for her sake.
As time went on, months following months till they grew

into years, while the amount down to Archie's name at the
Savings Bank grew also, Archie became very grateful for
the wise advice of Richard Dunn.
For, after all, though he and Nannie waited years for

their wedding day, they did not wait so long as must have
been the case, if Archie had not persistently from the first
put aside every little sum he could spare from present
needs. Sometimes it was a few shillings, sometimes only a
few pence. But "many a little makes a mickle," says the old
proverb. Archie proved the truth of this proverb.
Little more need be said, except that when the wedding

did take place, Annie Wilmot was present, besides many
other kind friends. The wedding cake was a present from
Annie, as well as a beautiful family Bible, bound in morocco.
Nannie made a sweet-looking young bride, in her neat
brown dress and bonnet; and even Nannie's own parents
scarcely saw her with more of fond pride than did Archie's
mother. For Mrs. Stuart had long ceased to regret the
thought of Nannie becoming Archie's wife; and on that
wedding day, she gained a "daughter indeed" to be the
comfort of her old age.
THE END.
Printed by BALLANTYNE, HANSON & CO.
Edinburgh & London

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PDF Regulated Cell Death Part A Apoptotic Mechanisms 1st Edition Avi Ashkenazi Download

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Regulated cell death Part A apoptotic mechanisms

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Hydrogen Peroxide and Cell Signaling Part C 1st Edition

JOHN N. ABELSON and MELVIN I. SIMON

ANNA MARIE PYLE

SIDNEY P. COLOWICK and NATHAN O. KAPLAN

First edition 2014

Copyright © 2014, Elsevier Inc. All Rights Reserved.

For information on all Academic Press publications

*Present address: Biotechnology Department, MS Ramaiah Institute of Technology, Bangalore, India.

Cell turnover is a fundamental feature of metazoan biology. Severe damage

Examining the Molecular

Methods in Enzymology, Volume 544 # 2014 Elsevier Inc. 1

regulation of membrane permeabilization by Bcl-2 family proteins. Here, we discuss four

2. AN IN VITRO FLUORESCENCE-BASED LIPOSOME

mimicking that of the cytoplasm and the interior of cellular organelles. We

2.1. Expression and purification of Bcl-2 family proteins

2.1.2 Purification of Bax and Bcl-XL

2.1.3 Purification of cBid

2.1.4 Buffer recipes

2.2. Site-specific protein labeling

attached thiol reactive moieties such as iodoacetamide or maleimide deriv-

3. Absorbance spectroscopy is used to determine protein containing frac-

2.3. Production of mitochondria-like liposomes

while preserving their authentic functions. It is possible to more directly

2.3.1 Preparing lipid films and generating liposomes

Table 1.1 Mitochondria-like lipid film composition

the association of the hydrophobic tails, forming the center of the

3. MEMBRANE PERMEABILIZATION ASSAY

3.1. ANTS/DPX release assay

ANTS/DPX in solution (Billen et al., 2008; Yethon et al., 2003). Frac-

4. FLUORESCENCE RESONANCE ENERGY TRANSFER

4.1. Detecting the interaction between two proteins

5. TRACKING THE CONFORMATION CHANGES

5.1. NBD-emission assay

Both residues 3 and 175 of Bax transition to a more hydrophobic environ-

environment change of the residue can be tracked over time, kinetics of

6. DETERMINING THE TOPOLOGY OF PROTEINS

6.1. Iodide quenching of NBD-labeled Bax

oxidation) and potassium chloride (2 M) stock solutions are added to

Photoreactive Stapled Peptides

Methods in Enzymology, Volume 544 # 2014 Elsevier Inc. 25

A. Cell surface B. Plasma membrane

C. Cytosol and mitochondrion

member-specific communication. The canonical complex between a

Multi-BH domain Multi-BH domain BH3-only

Heavy cell stress Weak cell stress

BAX/BAK activation Anti-apoptotic BH3-only displacement Anti-apoptotic Anti-apoptotic BAX/BAK

Cell death Cell survival

alpha-helical peptides for use as research tools and prototype therapeutics

2. OVERVIEW OF PHOTOREACTIVE STABILIZED