biophys_tech_

BI88CH02_Dobson ARjats.
cls May 22, 2019 10:47
Annual Review of Biochemistry

Biophysical Techniques in
Structural Biology
Access provided by Indian Institute of Technology - Mumbai/ Bombay on 05/09/23. For personal use only.
Christopher M. Dobson
Centre for Misfolding Diseases, Department of Chemistry, University of Cambridge,
Annu. Rev. Biochem. 2019.88:25-33. Downloaded from www.annualreviews.org
Cambridge CB2 1EW, United Kingdom; email: [email protected]
Annu. Rev. Biochem. 2019. 88:25–33 Keywords

First published as a Review in Advance on
biophysical techniques, mass spectrometry, MS, integrative structure
April 15, 2019
modeling, X-ray crystallography, cryo–electron microscopy, cryo-EM,
The Annual Review of Biochemistry is online at
X-ray free-electron laser, XFEL, time-resolved structural studies
biochem.annualreviews.org
https://doi.org/10.1146/annurev-biochem-013118- Abstract
111947
Over the past six decades, steadily increasing progress in the application of
Copyright © 2019 by Annual Reviews.
the principles and techniques of the physical sciences to the study of biolog-
All rights reserved
ical systems has led to remarkable insights into the molecular basis of life.
Of particular significance has been the way in which the determination of
the structures and dynamical properties of proteins and nucleic acids has so
often led directly to a profound understanding of the nature and mechanism
of their functional roles. The increasing number and power of experimental
and theoretical techniques that can be applied successfully to living systems
is now ushering in a new era of structural biology that is leading to fun-
damentally new information about the maintenance of health, the origins
of disease, and the development of effective strategies for therapeutic inter-
vention. This article provides a brief overview of some of the most powerful
biophysical methods in use today, along with references that provide more
detailed information about recent applications of each of them.
In addition, this article acts as an introduction to four authoritative re-
views in this volume. The first shows the ways that a multiplicity of bio-
physical methods can be combined with computational techniques to define
the architectures of complex biological systems, such as those involving weak
interactions within ensembles of molecular components. The second illus-
trates one aspect of this general approach by describing how recent advances
in mass spectrometry, particularly in combination with other techniques, can
generate fundamentally new insights into the properties of membrane pro-
teins and their functional interactions with lipid molecules. The third review
25
BI88CH02_Dobson ARjats.cls May 22, 2019 10:47
demonstrates the increasing power of rapidly evolving diffraction techniques, employing the very
short bursts of X-rays of extremely high intensity that are now accessible as a result of the con-
struction of free-electron lasers, in particular to carry out time-resolved studies of biochemical
reactions. The fourth describes in detail the application of such approaches to probe the mech-
anism of the light-induced changes associated with bacteriorhodopsin’s ability to convert light
energy into chemical energy.
Contents
THE EMERGENCE OF STRUCTURAL BIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

THE DEVELOPMENT OF A MULTIPLICITY OF BIOPHYSICAL
TECHNIQUES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
THE DYNAMICS AND HETEROGENEITY OF MACROMOLECULAR

STRUCTURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
THE CHARACTERIZATION OF MACROMOLECULAR INTERACTIONS . . . 28
REVOLUTIONS IN X-RAY CRYSTALLOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
LOOKING TO THE FUTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
THE EMERGENCE OF STRUCTURAL BIOLOGY

In 1951, Linus Pauling and Robert Corey (1) proposed α-helices and β-sheets as the fundamental
elements of the organization of polypeptide chains within protein molecules. Shortly afterward,
James Watson and Francis Crick (2) described the double-helical architecture of DNA, an achieve-
ment that led immediately to an understanding of the way that genetic information is stored and
passed on to future generations. Some five years later, the first structure of a protein, that of myo-
globin, was defined, revealing extensive helices of exactly the type predicted by Pauling and Corey
and indicating that proteins have specific global folds, a crucial step in the development of our
knowledge of their nature and properties (3). The structure of hemoglobin at increasing levels of
resolution followed soon afterward and led to our understanding of the manner in which oxygen
is transported though the bloodstream, along with many other aspects of its physiological role
(4). Indeed, this work also led to the first insights into the molecular origins of a medical condi-
tion linked to an aberrant conformation of a protein, in this case sickle cell disease (5), an area of
research that is now of major importance in the context of the increasing prevalence of neurode-
generative disorders (6). The determination of the structure of lysozyme in the mid-1960s then
provided the first view of an enzyme at atomic resolution (7) and led very rapidly to studies of
its complexes with substrate analogs, leading to a proposal for its catalytic mechanism (8). These
early demonstrations that a knowledge of molecular structure can reveal so clearly the nature of
biological function led to the recognition of the huge potential of the emerging field of structural
biology.
All these seismic advances were made possible as a result of the formulation of Bragg’s law
in 1913, leading to the ability to use the diffraction of X-rays to determine structures, initially
those of simple inorganic salts (9). It is fascinating to realize that Lawrence Bragg, who with his
father William shared the Nobel Prize for this work, was the Director of the Royal Institution
in London when the structure of lysozyme was determined in 1965. Remarkably, exactly 50 years
after receiving the Nobel Prize for defining the principles of X-ray diffraction, Bragg himself
26 Dobson
sketched out the overall fold of the first enzyme structure to be visualized by their application;
indeed, a version of this drawing, made from his careful observation of the electron density map,
appears in the original publication (7). These events exemplify how the discovery of a physical
technique can slowly but surely lead to its application to biological systems and provide deep
insight into the mechanisms of fundamental biological processes. Since these early applications to
nucleic acids and proteins, X-ray crystallography has generated many thousands of structures of
increasingly intricate molecules and complexes. One recent example is that of the ribosome, whose
high-resolution structure has provided a new level of understanding of protein biosynthesis and
also of the mechanism of action of many antibiotics, information that is leading to the rational
design of much-needed new drugs to combat resistant pathogenic organisms (10).
THE DEVELOPMENT OF A MULTIPLICITY OF BIOPHYSICAL

TECHNIQUES
This process of the translation of methods and ideas from physics to biology, creating the disci-
pline of biophysics, has been replicated with many other techniques—for example, electron mi-
croscopy, NMR spectroscopy, mass spectrometry (MS), and a range of optical spectroscopies in-
cluding circular dichroism and multiple types of fluorescence measurements. The methodological
developments in each of these techniques, as in X-ray crystallography, have been dramatic, and
their effects on structural biology have been transformative; an appreciation of the range of some
current applications of such methods can be gained from a selection of recent articles in Annual
Reviews journals. Thus, for example, cryo–electron microscopy (cryo-EM) can now generate the
structures of even very large molecules and complexes at atomic resolution without the need for
crystallization; for many years it was considered to be impossible to achieve such resolution, but
as so often is the case, a combination of scientific intuition and new technology has generated
major advances (11). NMR spectroscopy has made possible the determination of macromolecular
structures in the solution environments in which they function, and the use of specialized solid-
state techniques has made it possible to probe their conformations in other environments, such as
membranes (12) and pathological aggregates (13). MS has now advanced to the stage where the
structural properties of large biological complexes can be studied in their functional environments
(14). Optical techniques, particularly those involving fluorescence, have also made huge advances,
enabling them to be applied at the single-molecule level (15) and to generate images at much
higher resolution than was previously considered possible; the assumption that optical methods
could not provide information about structural features smaller than the wavelength of light has
been shown to be incorrect through the development of super-resolution techniques (16).
Shortly after the determination of the first structures by X-ray diffraction, it was recognized
that proteins with high levels of sequence similarity were likely to have similar structures. In-
deed, a very early example of molecular homology modeling was the generation of a model of
α-lactalbumin from that of lysozyme, whose function is completely different but whose sequence
is very similar (17); the good agreement between this model and the experimental structure when
it was determined confirmed the viability of this approach. Since then, the huge increase in the
number of structures in the Protein Data Bank and of protein sequences in UniProt has made it
possible to understand in increasing detail the relationships between sequence and structure, hence
allowing the modeling of many unknown structures and indeed the design of novel amino acid se-
quences that fold into specific structures (18). All these opportunities have benefitted hugely from
advances in instrumentation, ranging from the construction of ever more powerful X-ray sources
to the development of high-throughput sequencing centers. In addition, the rapid and contin-
uing increases in computer power have played a vital part in all these developments, as in the
www.annualreviews.org • Biophysical Techniques in Structural Biology 27

application of simulation techniques that give insight into the properties of macromolecules that
are challenging to define by experiment, such as many aspects of dynamic behavior and reaction
mechanisms (19).
THE DYNAMICS AND HETEROGENEITY OF MACROMOLECULAR

STRUCTURES
The recognition that the structural characterization of biological macromolecules also involves a
definition of their dynamical properties was one of the very interesting, and initially surprising,
results of the characterization of increasing numbers of biological macromolecules at high res-
olution. Indeed, early NMR studies of proteins revealed that fluctuations occur within even the
well-defined interiors of their apparently highly ordered structures, and molecular dynamics sim-
ulations showed that protein structures are undergoing a wide range of different types of motion
on different timescales and of different magnitudes (20). It is also now evident that many pro-
teins contain intrinsically disordered regions in their structures, or in some cases do not adopt any
persistent structure except when complexed to other molecules (21). Moreover, it soon became
evident that at least some of the dynamical properties of macromolecules are of vital importance
for their functional roles. Indeed, the pioneering studies of hemoglobin showed the critical ability
to convert between different conformational states in its mechanism of action, thereby enabling
much more efficient uptake and release of oxygen than would be possible with a rigid structure
(22). Such dynamical events have increasingly been found to be vital in many different types of
biological processes, ranging from enzymatic catalysis to the action of molecular motors such as
the kinesins and myosins involved in controlling complex biological functions in multiple forms
of life (23).
The existence of extensive dynamical behavior and of multiple conformations adds signifi-
cant challenges to the determination of the high-resolution structures of macromolecules in spe-
cific functional states. Moreover, the evident importance of the dynamical nature of biological
molecules for their mechanism of action has led to the conclusion that more complete descrip-
tions of their structures and functions could result from both the use of a concerted application
of multiple experimental techniques that can provide complementary information and a combi-
nation of experimental and computational methods. The review in this volume by Braitbard et al.
(24) provides an overview of these approaches under the title of “Integrative Structure Modeling.”
The article is focused on the ways that structural information can be obtained about systems that
have not succumbed to high-resolution analysis at the atomic level by conventional approaches,
particularly X-ray crystallography and NMR spectroscopy, because of their failure to crystallize,
or their size, or their heterogeneity as a result of a multiplicity of different conformational states.
The first section of this article describes many of the biophysical techniques that are most widely
used for this purpose, in particular, methods such as cross-linking combined with mass spectrom-
etry (XL-MS), small-angle X-ray scattering (SAXS), Förster resonance energy transfer (FRET)
between fluorophores, and cryo-EM. It then describes how such techniques can be combined,
with the aid of molecular modeling approaches, to provide information about the structures of
even very large complexes of proteins and other molecules, at least at the level of their overall
architecture, that would not otherwise be accessible (24).
THE CHARACTERIZATION OF MACROMOLECULAR INTERACTIONS

Braitbard et al. (24) also discuss the fact that the resolution revolution in cryo-EM has resulted in
the determination of electron density maps obtained from single-particle analysis at resolutions
28 Dobson
below 4 Å, compared with resolutions of 20–30 Å that were the best that could be achieved just
a few years ago (11). Some of the problems that required integrative structure modeling in the
past can now be addressed rapidly and efficiently by cryo-EM. In other cases—for example, those
for which it is hard to isolate intact complexes or, indeed, for relatively small particles—cryo-EM
may be possible only at much lower resolution, and such modeling approaches become of great
importance. The exciting developments through which cryo–electron tomography (25) can pro-
vide images of whole cells in three dimensions is another area where structural modeling can play
a key role. In addition, other new approaches, such as those based on microfluidic techniques (26),
are increasingly able to characterize even weak interactions between macromolecules in environ-
ments that mimic those within biological systems and hence provide information for the modeling
of transient complexes. In the final part of their article, Braitbard et al. (24) discuss the types of
scoring functions that are vital for assessing the most probable models that can be derived from
the combination of structural data obtained by the different methods, and they then provide a
range of case studies in which high-resolution structures have subsequently been determined, in
particular by cryo-EM, to assess their accuracy. The results show that the integrative approach is
generally very successful in defining the overall architecture of even highly complex assemblies,
although some aspects of the structures, such as the orientation of specific component molecules,
can be harder to define (24).
The manner in which advances in a specific biophysical method can open up new opportunities
to extend our understanding of the nature and significance of the interactions of biological macro-
molecules in complex environments is discussed in the review by Bolla et al. (27) in the context of
membrane proteins. In particular, this review describes the remarkable developments in MS that
have taken place in recent years, such that it is now possible, by exploiting a range of soft ioniza-
tion processes, to maintain macromolecules and their complexes in native-like environments after
projection into the gas phase, an approach described as native MS. A range of sophisticated exper-
iments can then be carried out to probe in detail key aspects of their structures and interactions
through measurements of their mass, shape, and stability (14). The information obtained in this
manner can be combined, in ways related to those discussed by Braitbard et al. (24), with that from
other methods, notably X-ray crystallography and cryo-EM, to provide detailed descriptions of
even highly complex and dynamic systems.
Bolla et al. (27) focus particularly on the investigation of the lipid molecules that surround
membrane proteins in their cellular environments and are major determinants of their structural
properties and their functional roles. The key developments in such studies involve the use of
membrane mimetics that are able to preserve the integrity of the native structure of the protein
concerned and to maintain ligand interactions that are associated with its functional role. Cur-
rently, the most widely used membrane mimetics in native MS are a variety of types of detergent
micelles (27) that can be optimized to enable the study of specific types of membrane protein.
Many other systems, however, are being developed for solubilizing membrane proteins free of de-
tergents, including lipid vesicles; nanodiscs, consisting of phospholipids and membrane scaffold
proteins; and SMALPs (styrene-maleic acid lipid particles), in which membrane proteins are sur-
rounded by lipid bilayers covered by a styrene-maleic acid (SMA) copolymer. Bolla et al. (27) give
a comprehensive summary of the results of the present applications of native MS to probe the
interactions of membrane proteins with lipids that are crucial for their structure and function, in-
cluding channels, efflux pumps, kinases, and receptors. As membrane mimetics become ever more
sophisticated, native MS, particularly when combined with other biophysical techniques, has the
potential to provide deep insights into the structural features and intermolecular interactions that
define the functional properties of membrane proteins.

REVOLUTIONS IN X-RAY CRYSTALLOGRAPHY

Diffraction techniques initiated the field of structural biology and remain as a crucial foundation
of this whole area of science. Indeed, their power was hugely increased by the application to pro-
tein crystallography, beginning in the 1970s, of synchrotron radiation sources that can provide
much higher intensity beams of X-rays (28). Although the extent of radiation damage, whereby
the molecules with which the radiation interacts are disrupted or destroyed, increases as the beam
intensity is increased, reduction in the temperature of the samples can cause this phenomenon to
be greatly reduced; similar factors for electron beams are the reason for the increased power of
cryo-EM over conventional electron microscopy. It became increasingly evident, however, that
radiation damage can also be greatly limited if very short bursts of X-rays are used. For structural
studies, it was apparent that such bursts would need to be of extremely high intensity if useful
diffraction data were to be obtained for macromolecular samples. Such bursts are not available
from the storage rings of synchrotrons but can be obtained from the linear accelerators of X-ray
free-electron lasers (XFELs), which can deliver a gain of up to nine orders of magnitude in bright-
ness for periods of tens of femtoseconds (fs) (10−15 s); such times are then short enough to limit
sufficiently the structural changes associated with even these intense beams of radiation to allow
diffraction studies to be carried out (29).
The review in this volume by Chapman (30) provides fascinating details of the developments
that have led to the recent application of XFELs to problems in structural biology. It points out that
an initial motivation for the use of XFELs was to carry out diffraction studies on single molecules
and complexes without the need for crystallization, in much the way that single-particle imaging
is now possible using cryo-EM. Although such studies have advanced significantly, and have been
shown to be applicable in principle through experiments with virus particles, they are not yet gen-
erally viable. These ideas have, however, enabled high-resolution structures of macromolecules in
small crystals to be determined without the need for cryogenic cooling and have led to the devel-
opment of time-resolved studies of conformational dynamics and of intermediates formed during
chemical reactions, such as those of enzymes (30). This approach opens the door to studies of the
mechanisms of biological process taking place at physiological temperatures and on timescales
ranging from 100 fs to minutes. To carry out such experiments, it is necessary to collect extensive
data sets but to avoid cumulative radiation damage, and highly innovative procedures have been
devised for this purpose (31). Thus, for example, samples of liquids containing vast numbers of
microcrystals in which a reaction has been initiated (e.g., by a burst of light) can be injected into
the electron beam, allowing sufficient data to be collected at a given time point for the structure
to be defined, and then again at subsequent time points, enabling the structural changes taking
place during the reaction to be monitored.
This method has already proved to be viable and indeed to be very powerful (32). In addition
to ensuring that the molecules in the crystalline samples are still bathed in liquid under defined
conditions, it appears too that conformational changes taking place during a reaction are less
restrained in microcrystals than in larger assemblies, probably simply because more of the sample
is close to the surface of the crystalline array. As well as describing such approaches that have
already been developed, Chapman (30) discusses the advances that will be needed, and are already
emerging, to increase throughput and hence to probe the effects of a wide range of variables so
as to give deeper insights into even complex mechanisms. In addition, he outlines the types of
further advances that will be needed if this type of experiment is to be routinely applicable to
single particles, hence eliminating the need for sample crystallization.
In the fourth article in this set of themed reviews, Wickstrand et al. (33) focus on the use
of XFEL techniques for time-resolved studies of a specific biological system, that of bacteri-
orhodopsin (bR), the archetypal example of a light-driven proton pump, and then describe the
30 Dobson
results of such studies with those from intermediate trapping, in which the reaction is triggered in
crystals and subsequently quenched at different time points to allow their study by conventional
synchrotron X-ray crystallography. The history of structural studies of bR illustrates very clearly
the advances in structural biology over the last half century in the context of membrane proteins.
Thus, as Wickstrand et al. (33) describe, in 1975 electron diffraction techniques generated a low-
resolution view of the molecule and revealed the transmembrane helices that are a characteristic
feature of bR and many other membrane proteins, another milestone in the progress of structural
biology. In 1990, the first atomic-resolution model of bR was determined, again using electron
diffraction, followed by structures from X-ray diffraction studies of crystals generated by innova-
tive methods developed specifically for membrane proteins. This advance led to the beginnings of
the study of the time dependence of light-induced structural changes in bR by intermediate trap-
ping methods, revealing information about the different states that become populated at different
times after illumination.
A large number of studies of bR using this approach have been reported over the intervening
years, and indeed some of the conclusions from them were initially controversial. The appli-
cation of time-resolved serial femtosecond crystallography (TR-SFX) has, however, enabled
the intermediate trapping experiments at low temperature to be correlated with measurements
at room temperature, and indeed many of the conclusions of these earlier studies have been
confirmed and then defined in greater detail by TR-SFX (33). Although it is possible that some
large conformational changes in the molecule are not observed in either approach because of the
constraints of the crystalline environment, the results enable further details of the mechanisms
of the multiple events taking place over timescales differing in orders of magnitude to be defined,
generating increasingly profound insights into the means by which light energy is ultimately
converted into chemical energy.
LOOKING TO THE FUTURE

These four reviews illustrate two very important aspects of the development of physical methods
for applications in biology. On the one hand, individual techniques become ever more capable of
providing high-resolution information about complex structures and dynamics, and on the other
hand, the ability to integrate information from a multitude of lower-resolution biophysical meth-
ods enables descriptions of less-well-defined systems with high levels of heterogeneity and exten-
sive dynamical character to be achieved, at least at the level of their overall architectures. Indeed,
the combination of complementary experimental and theoretical biophysical approaches is un-
doubtedly the key to achieving a molecular understanding of the functional mechanisms of many
increasingly complex biological systems. On a broader front, these articles illustrate the remark-
able coming of age of biophysical methods over the past few decades, ushering in a new golden
age of structural biology that is providing unprecedented insights into our understanding of the
multitude of interactions between molecules in their cellular environments. Comparison of the
nature and regulation of these interactions in health and disease will undoubtedly contribute to
developments in health care, drug discovery, and therapeutic strategies that have the potential to
transform our lives in the years to come.
DISCLOSURE STATEMENT
The author is not aware of any affiliations, memberships, funding, or financial holdings that might
be perceived as affecting the objectivity of this review.

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BI88_FrontMatter ARI 22 May 2019 14:41
Annual Review of
Biochemistry
Contents Volume 88, 2019
Moving Through Barriers in Science and Life

Judith P. Klinman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Biophysical Techniques in Structural Biology

Christopher M. Dobson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p25
X-Ray Free-Electron Lasers for the Structure and Dynamics of

Macromolecules
Henry N. Chapman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p35
Bacteriorhodopsin: Structural Insights Revealed Using X-Ray Lasers
and Synchrotron Radiation
Cecelia Wickstrand, Przemyslaw Nogly, Eriko Nango, So Iwata, Jörg Standfuss,
and Richard Neutze p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p59
Membrane Protein–Lipid Interactions Probed Using Mass
Spectrometry
Jani Reddy Bolla, Mark T. Agasid, Shahid Mehmood, and Carol V. Robinson p p p p p p p p p p p85
Integrative Structure Modeling: Overview and Assessment
Merav Braitbard, Dina Schneidman-Duhovny, and Nir Kalisman p p p p p p p p p p p p p p p p p p p p p 113
Eukaryotic Base Excision Repair: New Approaches Shine Light on
Mechanism
William A. Beard, Julie K. Horton, Rajendra Prasad, and Samuel H. Wilson p p p p p p p p p 137
Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor
in Repair and Replication
Jacqueline K. Barton, Rebekah M.B. Silva, and Elizabeth O’Brien p p p p p p p p p p p p p p p p p p p p p p 163
Evaluating and Enhancing Target Specificity of Gene-Editing
Nucleases and Deaminases
Daesik Kim, Kevin Luk, Scot A. Wolfe, and Jin-Soo Kim p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 191
The BRCA Tumor Suppressor Network in Chromosome Damage
Repair by Homologous Recombination
Weixing Zhao, Claudia Wiese, Youngho Kwon, Robert Hromas,
and Patrick Sung p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221
Cancer Treatment in the Genomic Era
Gary J. Doherty, Michele Petruzzelli, Emma Beddowes, Saif S. Ahmad,
Carlos Caldas, and Richard J. Gilbertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 247
v
Eukaryotic Ribosome Assembly

Jochen Baßler and Ed Hurt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 281
The Organizing Principles of Eukaryotic Ribosome Recruitment
Jerry Pelletier and Nahum Sonenberg p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 307
Mechanisms of Cotranslational Maturation of Newly Synthesized
Proteins
Günter Kramer, Ayala Shiber, and Bernd Bukau p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 337
Lysine-Targeted Inhibitors and Chemoproteomic Probes
Adolfo Cuesta and Jack Taunton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 365

Horizontal Cell Biology: Monitoring Global Changes of Protein
Interaction States with the Proteome-Wide Cellular Thermal Shift
Assay (CETSA)
Lingyun Dai, Nayana Prabhu, Liang Ying Yu, Smaranda Bacanu,
Anderson Daniel Ramos, and Pär Nordlund p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 383
Soluble Methane Monooxygenase
Rahul Banerjee, Jason C. Jones, and John D. Lipscomb p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 409
Glycoengineering of Antibodies for Modulating Functions
Lai-Xi Wang, Xin Tong, Chao Li, John P. Giddens, and Tiezheng Li p p p p p p p p p p p p p p p p p 433
Lysosomal Glycosphingolipid Storage Diseases
Bernadette Breiden and Konrad Sandhoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 461
Exosomes
D. Michiel Pegtel and Stephen J. Gould p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 487
Structure and Mechanisms of F-Type ATP Synthases
Werner Kühlbrandt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 515
ECF-Type ATP-Binding Cassette Transporters
S. Rempel, W.K. Stanek, and D.J. Slotboom p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 551
The Hippo Pathway: Biology and Pathophysiology
Shenghong Ma, Zhipeng Meng, Rui Chen, and Kun-Liang Guan p p p p p p p p p p p p p p p p p p p p p p 577
Small-Molecule-Based Fluorescent Sensors for Selective Detection of
Reactive Oxygen Species in Biological Systems
Xiaoyu Bai, Kenneth King-Hei Ng, Jun Jacob Hu, Sen Ye, and Dan Yang p p p p p p p p p p p p 605
Single-Molecule Kinetics in Living Cells
Johan Elf and Irmeli Barkefors p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 635
Molecular Mechanism of Cytokinesis
Thomas D. Pollard and Ben O’Shaughnessy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 661
Mechanism and Regulation of Centriole and Cilium Biogenesis
David K. Breslow and Andrew J. Holland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 691
vi Contents
The Structure of the Nuclear Pore Complex (An Update)

Daniel H. Lin and André Hoelz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 725
Propagation of Protein Aggregation in Neurodegenerative Diseases
Jaime Vaquer-Alicea and Marc I. Diamond p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 785
Botulinum and Tetanus Neurotoxins
Min Dong, Geoffrey Masuyer, and Pål Stenmark p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 811
Errata
An online log of corrections to Annual Review of Biochemistry articles may be found at

http://www.annualreviews.org/errata/biochem
Contents vii

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BI88CH02_Dobson ARjats.

cls May 22, 2019 10:47

Annual Review of Biochemistry

Cambridge CB2 1EW, United Kingdom; email: [email protected]

Annu. Rev. Biochem. 2019. 88:25–33 Keywords

THE EMERGENCE OF STRUCTURAL BIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

THE DYNAMICS AND HETEROGENEITY OF MACROMOLECULAR

THE EMERGENCE OF STRUCTURAL BIOLOGY

THE DEVELOPMENT OF A MULTIPLICITY OF BIOPHYSICAL

www.annualreviews.org • Biophysical Techniques in Structural Biology 27

THE DYNAMICS AND HETEROGENEITY OF MACROMOLECULAR

THE CHARACTERIZATION OF MACROMOLECULAR INTERACTIONS

www.annualreviews.org • Biophysical Techniques in Structural Biology 29

REVOLUTIONS IN X-RAY CRYSTALLOGRAPHY

LOOKING TO THE FUTURE

www.annualreviews.org • Biophysical Techniques in Structural Biology 31

hemoglobin fibers. PNAS 70:718–22

www.annualreviews.org • Biophysical Techniques in Structural Biology 33

Moving Through Barriers in Science and Life

Biophysical Techniques in Structural Biology

X-Ray Free-Electron Lasers for the Structure and Dynamics of

Eukaryotic Ribosome Assembly

Adolfo Cuesta and Jack Taunton p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 365

The Structure of the Nuclear Pore Complex (An Update)

An online log of corrections to Annual Review of Biochemistry articles may be found at

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