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trends

Applications of circular dichroism in

protein and peptide analysis
Norma J. Greeneld

UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA
This review discusses several useful applications of circular dichroism as a tool for analyzing properties of proteins. The following
topics are discussed: ( 1 ) protein^ligand interactions; ( 2 ) thermodynamics of protein folding; ( 3 ) conformational transitions and protein aggregation; ( 4 ) folding intermediates;
( 5 ) kinetics of protein folding. Specic examples are given to illustrate each application. z1999 Elsevier Science B.V. All rights
reserved.
Keywords: Circular dichroism; Protein-folding;
Protein^ligand binding; Thermodynamics

1. Introduction
Circular dichroism ( CD ) is a valuable spectroscopic technique for studying protein structure in solution because many common conformational motifs,
including K-helixes, L-pleated sheets, poly-L-proline
II-like helices and turns, have characteristic far UV
( 178^250 nm ) CD spectra. For example, K-helices
display large CD bands with negative ellipticity at
222 and 208 nm, and positive ellipticity at 193 nm,
L-sheets exhibit a broad negative band near 218 nm
and a large positive band near 195 nm, while disordered extended chains have a weak broad positive CD
band near 217 nm and a large negative band near 200
nm. The spectrum of a protein is basically the sum of
the spectra of its conformational elements, and thus
CD can be used to estimate secondary structure. In
addition, the chromophores of the aromatic amino
acid proteins are often in very dissymmetric environments resulting in distinctive CD spectra in the near
UV ( 250^300 nm ), which can serve as useful probes
of protein tertiary structure. Recently several articles
[ 1^3 ] have compared and evaluated most of the currently available computer methods for analyzing CD

spectra to obtain the secondary structure of proteins,

and thus this topic will not be discussed here.
Circular dichroism, however, has many more facets
than simply being a tool to estimate protein structure.
For example, it is an excellent technique for determining the thermodynamics and kinetics of protein folding and denaturation and is unsurpassed for following
the effects of mutations on protein folding and stability. In addition, it can be an excellent tool for following
protein^ligand interactions. As sophisticated mathematical programs for tting nonlinear equations and
deconvoluting sets of curves using personal computers have become increasingly available, the analysis of thermodynamic and kinetic experiments, which
can be performed by following changes in CD spectra,
have become much simpler, increasing the usefulness
of the technique.
It would be impossible to do a complete review of
all of the recent applications involving CD spectroscopy. For example, in the last 10 years there have been
more than 1500 papers published in the Journal of
Biological Chemistry alone, where CD was used to
study various aspects of protein and nucleic acid
chemistry. This review will therefore focus on several
specic applications that illustrate of the general usefulness of CD as an analytical tool. The following
topics will be discussed: ( 1 ) protein^ligand interactions; ( 2 ) thermodynamics of protein folding; ( 3 )
conformational transitions and protein aggregation
phenomena; ( 4 ) folding intermediates; and ( 5 )
kinetics of protein folding. Specic examples will be
used to illustrate each application.

2. Protein^ligand interactions
Circular dichroism can be used to follow protein^
ligand interactions and protein denaturation because
CD is a quantitative technique. Like other optical
measurements, CD measurements obey Beer's law:

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1999 Elsevier Science B.V. All rights reserved.

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trends in analytical chemistry, vol. 18, no. 4, 1999

the CD spectrum of each component in a solution is

directly proportional to its concentration, and the total
spectrum represents the sum of all the contributing
spectra. Thus, the change in the CD spectrum of a
protein upon addition of a ligand, denaturant or heat
is directly proportional to the amount of protein
changed by the perturbation.
Circular dichroism has been used to study protein^
ligand interactions since the instrumentation rst
became available in the late 1960s [ 4 ]. Circular
dichroism can be used to follow the binding of ligands
to proteins, peptides and nucleic acids provided one of
two criteria is fullled. Either the binding of ligand
must bind in an dissymmetric fashion that induces
extrinsic optical activity in the chromophores of the
bound ligand, or the binding must result in a conformational change in the macromolecule that results in a
change in its intrinsic CD spectrum. Before efcient
algorithms for solving nonlinear equations became
widely available, it was usually necessary to linearize
the binding equations to obtain binding constants from
CD data ( reviewed in [ 5 ]). Today it is usual to t the
nonlinear binding equations directly using commercial programs such as SigmaPlot0 , Origin0 , GraphPad0 and PsiPlot0 , among others, which use tting
techniques such as the Levenberg^Marquardt algorithm [ 6 ] to estimate the constants which best t the
data.
2.1. Extrinsic effects

When highly chromatic ligands bind to proteins

they may generate large CD bands, called Cotton
effects, in the region of their absorbance spectrum
if they bind in a dissymmetric fashion. The change
in ellipticity as a function of substrate concentration can be used to estimate the binding constants.
For example, assume a ligand with concentration
[ Li ] binds to a protein with a concentration [Po ],
with n equivalent binding sites where [ LPi ] is the
concentration of protein^ligand complex formed.
The equilibrium constant, K, obeys the following
equation:
K LPi =Li 3nLPi Po 3LPi

The change in ellipticity, ai , due to complex formation

is directly proportional to [ LPi ]. When all of the protein has bound ligand, ai = amax
LPi Po ai =amax

Fig. 1. ( A ) The molar circular dichroism of dihydrofolate

reductase and 1:1 complexes with dihydrofolate ( dashed
line ), folate ( dot-dashed line ) and methotrexate ( solid
line ). ( B ) The increase in ellipticity at 292.5 nm as a function of dihyrofolate concentration: ( solid line ) enzyme plus
dihydrofolate, ( dashed line ) dihydrofolate alone. Reprinted with permission from [ 7 ]; z ( 1972 ) American
Chemical Society.

ai amax f1 KPo =n KPo =2KPo

3f1 KLi =n KPo =2KPo 2
3Li =nPo g1=2 g

By tting Eq. 3 to the raw data, the binding constant

of the ligand for the protein and the number of binding
sites can be estimated. To illustrate, when folates and
nucleotides bind to dihydrofolate reductase large CD
bands are induced [ 7 ]. Fig. 1A shows the extrinsic
Cotton effects induced in the near UV when dihydrofolate, folate and methotrexate bind to dihydrofolate
reductase from Escherichia coli, and Fig. 1B shows
the change in ellipticity as a function of added dihy-

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trends in analytical chemistry, vol. 18, no. 4, 1999

drofolate. The data were used to determine a binding

constant of 1.1 WM.
As well as being used to study the interactions of
substrates and inhibitors with enzymes, extrinsic Cotton effects have been used to study protein^ligand
interactions in diverse systems. For example, they
have been used to study the binding of cofactors to
proteins including the binding of nucleotides to lactic
dehydrogenase [ 8 ], ATP to GroEL [ 9 ], pyridoxal
phosphate to tryptophan synthase [ 10 ] and the binding of retinol to interphotoreceptor retinol binding
protein [ 11 ]. In addition extrinsic CD bands have
been employed to study the interactions of drugs
with bovine serum albumin ( e.g. [ 12 ]) and the interactions of DNA binding proteins with DNA ( e.g.
[ 13 ]).
2.2. Intrinsic effects

The binding of ligands to proteins often results in

conformational transitions. The resultant change in the
intrinsic ellipticity of the backbone amides caused by
the conformational change can also be used to obtain
the ligand binding constants. For example, divalent
cations are effectors in a many biological systems
that often induce large conformational changes in target proteins. For example, Ca2 ion binding to the
muscle regulatory protein, troponin C, induces an
increase in ellipticity due to a stabilization of K-helical
regions of the protein. Johnson and Potter [ 14 ]
showed that CD could distinguish two classes of
Ca2 binding sites in troponin C, with high and low
afnity. Calcium binding to the low afnity site regulates muscle contraction. Fig. 2 illustrates how the
change in ellipticity of troponin C (TnC ) as a function
of free calcium concentration can be used to obtain the
calcium binding constants for the high and low afnity
sites, ka1 and ka2 [ 15 ]. Data obtained with skeletal
muscle TnC, TnC expressed in E. coli, and a mutant
TnC lacking a helical segment at the N-terminus ( the
N-helix ) are illustrated. Chelators that bind Ca2 were
used as buffering agents so that the free Ca2 concentration was xed at the desired concentrations. The
changes in ellipticity at 222 nm as a function of free
Ca2 concentration were normalized to a scale of 0 ( no
Ca2 ) to 1 ( saturating Ca2 ) and t to the sum of two
Hill equations:
H1
H1
ai a1 ka1
Ci H1 =1 ka1
Ci H1
H2
H2
a2 ka2
Ci H2 =1 ka2
Ci H2

Fig. 2. The increase in ellipticity of troponin C (TnC ) at 222

nn, due to increased K-helical content, as a function of free
Ca2 concentration. R, muscle TnC; a, wild type TnC
expressed in E. coli; b, recombinant TnC missing the
rst 14 N-terminal residues. Ellipticities at 222 nm are normalized to a scale of 0 ( no Ca2 ) to 1 ( saturating Ca2 ).
The points are the raw data, and the lines are the data tted
by the sum of two Hill equations. Reprinted with permission from [ 15 ]; z ( 1994 ) The American Society for Biochemistry and Molecular Biology, Inc.

where ai is the change in ellipticity at 222 nm observed

when calcium binds to TnC, H1 and H2 are the Hill
constants and a1 and a2 are the molar absorptivities for
the spectral change caused by calcium binding to the
high and low afnity sites, respectively, and [ Ci ] is the
free calcium concentration. Initial estimate of a1 , a2 ,
H1 , H2 , ka1 and ka2 were made and the equations were
t using the Levenberg^Marquardt algorithm [ 6 ]
implemented in the commercial program SigmaPlot.
The deletion of the N-helix caused a threefold decrease
in the binding of calcium to the regulatory site of TnC
[ 15 ].
There are many other classes of protein^ligand
interactions that result in conformational changes.
For example, DNA binding to DNA transcription factors results in an increase in helical content of the transcription factor. These factors often contain K-helical
coiled-coils or helix-loop-helix motifs, which serve as
dimerization domains, and a relatively unstructured
basic region which binds to DNA. Upon binding to
the DNA the basic region assumes a helical conformation. Fig. 3 illustrates the increase in ellipticity when
two muscle helix-loop-helix regulatory factors, E47
and MyoD bind to an oligonucleotide containing the
creatine enhancer site [ 16 ].
Another interesting class of protein^ligand interactions, resulting in conformational changes, is the bind-

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3. Thermodynamics of protein folding

Following changes in the optical activity of a protein or polypeptide as a function of temperature or
denaturant is a very convenient method of obtaining
the thermodynamics of folding. Indeed, the major
application of CD today is the determination of the
thermodynamics of protein folding and the effects of
mutations on protein stability. The equations describing the thermodynamics of structural stability and
cooperative folding behavior in proteins have been
reviewed by Freire [ 21 ]. The applications of CD to
studies of protein folding have been recently reviewed
by Kelly and Price [ 22 ].
3.1. Thermodynamic parameters of folding from
thermal denaturation and renaturation studies

Fig. 3. The backbone ellipticity of two muscle DNA transcription factors: a, E47 and E, MyoD, and O, a chimera,
MyoEL, in which the loop region of MyoD has been
replaced by that of E47. The CD spectra of all three proteins are similar and are characteristic of a partially helical
structure. Addition of an oligonucleotide containing the
creatine kinase enhancer site to b, E47 or F, MyoD
increases negative ellipticity at 222 and 208 nm characteristic of K-helices, because the basic region folds upon
binding to DNA. Reprinted with permission from [ 16 ];
z ( 1998 ) The American Society for Biochemistry and
Molecular Biology, Inc.

ing of heparin to diverse proteins including the aminoterminal domain of bronectin [ 17 ], hepatocyte
growth factor [ 18 ], the amyloid precursor protein of
Alzheimer's disease [ 19 ] and annexin [ 20 ] among
others. The binding of heparin has very different
effects, depending on its target. CD studies show
that a peptide derived from the amyloid precursor protein, APP416^447 shifts towards an K-helical conformation in the presence of heparin. In contrast, the
Ca2 -dependent binding of heparin to annexin caused
a large decrease in its K-helical content from approximately 44 to 31%, a small decrease in the L-sheet
content from approximately 27 to 24%, and an
increase in the unordered structure from 20 to 29%.
A peptide fragment corresponding to the heparin binding site of hepatocyte growth factor changes from a
random structure to a L-sheet-like structure upon heparin binding, which results in the oligomerization of
the growth factor.

Essentially the use of CD to follow protein denaturation depends on the fact that the change in ellipticity
is directly proportional to the change in concentration
of native and denatured forms. The observed ellipticity of a protein, aobs , changes when it unfolds. When
the protein is fully folded aobs = a1 and when it is fully
unfolded aobs = a2 . In the case of a monomeric protein,
the equilibrium constant of folding, K = folded /
unfolded. If we dene K as the fraction folded at a
given temperature then
K K=13K

vG nRTlnK

where vG is the free energy of folding and R is the gas

constant = 1.987 cal / mol.
vG vH3TvS

where vH is the enthalpy of folding and vS is the

entropy of folding. TM is the observed midpoint of
the thermal transition. At the TM , K = 1, therefore
vG = 0 and vS = vH /TM . Solving these equations we
obtain:
K expfvH=RTT=TM 31g

K K=1 K

aobs a1 3a2 K a2

10

To calculate the values of vH and TM that best

describe the folding curve, initial values of vH, TM ,

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3.2. Thermodynamic parameters of protein

folding using denaturants

Fig. 4. The change in fraction folded of GCN4 as a function

of temperature and peptide concentration. The concentrations range from 1 WM with the lowest TM to 20 WM with
the highest TM . The enthalpy of unfolding was 35.0 1.1
kcal / mol ( monomer ) which compared to that of 34.7 0.3
kcal / mol measured by calorimetry. Reprinted with permission from [ 25 ]; z ( 1993 ) American Chemical Society.

a1 and a2 are estimated, and Eq. 10 is tted to the

experimentally observed values of the change in ellipticity as a function of temperature, by a curve tting
routine such as the Levenberg^Marquardt algorithm
[ 6 ]. Since at K = 1, vG = 0, the entropy of folding can
therefore be calculated using Eq. 4 where vS = vH /
TM . The free energy of folding at any other temperature can then be calculated using Eq. 4. Note that this
treatment ignores changes in heat capacity, vCp , when
a protein unfolds, but equations have been developed
that include that term [ 23 ]. Similar equations can be
used to estimate the thermodynamics of folding of
proteins and peptides that undergo folded multimer
to unfolded monomer transitions ( reviewed in [ 24 ]).
The free energy of folding may also be calculating
from the concentration dependence of CD spectra, if
changes in association state are accompanied by
changes in conformation.
Fig. 4 illustrates how changes in ellipticity as a
function of temperature and concentration have been
used to determine the enthalpy of folding of the GCN4
transcription factor, which undergoes a two-state transition between a folded two-stranded K-helical coiledcoil and a monomeric disordered state [ 25 ]. The values for the enthalpy of the folding transition, determined from the changes in ellipticity as a function of
temperature agreed with the values obtained from
scanning calorimetry.

In some cases thermal folding and unfolding studies

are impractical. The protein may have a very high TM ,
or may aggregate and precipitate upon heating. In
these cases denaturants, such as urea or guanidineHCl, may be added to a protein or peptide to induce
unfolding. One obtains K at each concentration of
denaturant by assuming that the protein is native in
the absence of denaturant, and fully unfolded when
there is no further change in ellipticity upon addition
of higher concentrations of perturbant. In each case, K,
the equilibrium constant of folding is determined from

Fig. 5. ( A ) Concentration dependence of the guanidine

hydrochloride denaturation of a coiled-coil model peptide.
Concentrations of the peptide were b, 48 WM; O, 131 WM;
a, 213 WM; R, 592 WM; E, 888 WM and F, 1345 WM. ( B )
Plot of linear dependence of vG of unfolding on the concentration of guanidine hydrochloride at different concentrations of the peptide, extrapolated to zero to determine
the free energy of unfolding in the absence of denaturant.
The correlation coefcient for the regression line was 0.94.
Reprinted with permission from [ 26 ]; z ( 1992 ) The American Society for Biochemistry and Molecular Biology, Inc.

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Fig. 6. Time-dependent aggregation of islet amyloid polypeptide ( IAPP ) seeded with IAPP which had been subjected to
prolonged glycosylation to produce advanced glycosylation end products ( AGE ). After seeding the peptide went from a
conformation which was predominantly random with a small amount of K-helix to one with a high L-content, followed by
aggregation and precipitation out of solution. Reprinted with permission from [ 30 ]; z ( 1998 ) FEBS.

the ellipticity observed at each concentration of denaturant.

K K=nCn31 13Kn

11

where
K aobs 3a2 =a1 3a2

12

K, a1 and a2 have the same denitions as in the case of

thermal unfolding above. [ C ] is the total concentration of protein monomers. The free energy of folding is
evaluated at every concentration of denaturant using
the equation vG = 3nRTlnK. The free energies are
plotted as a function of denaturant and extrapolated
to zero denaturant to obtained the free energy of folding of the native material. Hodges and coworkers have
used this approach to study the contributions of hydrophobic and charged residues to the stability of
designed coiled-coil K-helices. Fig. 5 illustrates the
determination of the free energy of folding of a
coiled-coil model peptide from a guanidine denaturation study [ 26 ]. It should be noted that one may obtain
different apparent vGs of folding when different denaturants are employed because some denaturants, such
as solutions of guanidine-HCl, which have a high ionic
strength, disrupt salt bridges in proteins, while others,
such as urea, which is uncharged, have a lesser effect
on protein charge^charge interactions.

4. Conformational transitions
While CD is most usually used to study transitions
from a folded to an unfolded state, recently it has been
found that certain systems undergo transitions involving changes from an K-helical to a L-pleated sheet
conformation ( or vice versa ). Conformational transitions may be followed by oligomerization of the protein and / or aggregation and precipitation. Such conformational changes can have important clinical
consequences as it is felt that such transitions may
be involved in the pathology of diseases such as scrapie, `mad cow disease' and Alzheimer's disease. CD
has been used to follow conformational transitions in
prions ( e.g. [ 27 ]), the Alzheimer's L-amyloid peptide
( e.g. [ 28 ]) and amylin, an amyloid peptide seen in the
pancreatic islets in patients with diabetes ( e.g. [ 29 ]).
Fig. 6 illustrates conformational changes of islet amyloid polypeptide after seeding with peptide modied
with advanced glycosylation end products [ 30 ]. The
peptide rapidly goes from a conformation that is predominantly random, with a small amount of K-helix, to
a conformation with considerable L-structure. After
the structural transition, the peptide associates into
brils and precipitates out of solution.

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5. Folding intermediates
Often when proteins or peptides fold or unfold, the
process is not a two-state transition between the native
and totally disordered forms; there are intermediate
states. These states may be transient, and may be
observed during kinetic measurements of folding or
unfolding, or they may be stable intermediates, seen
when the peptide is subject to denaturing conditions,
such as high temperatures, or exposure to urea, guanidine or detergents. These states may be partially
folded, with dened tertiary structures, or may be so
called molten globules. The term molten globule was
rst introduced by Ptitsyn and coworkers ( for reviews
see [ 31,32 ]) to describe a compact state with nativelike secondary structure but slowly uctuating tertiary
structure.
CD can often be used to detect and quantify the
intermediate states. For example, some `molten globule' folding intermediates exhibit large CD signals in
the far UV arising from secondary structural elements
such as K-helices. The molten globule folding intermediates, however, display almost none of the near
UV CD bands arising from tertiary structural interactions of aromatic groups, which are often seen in the
spectra of native proteins. A classic example of a protein with a partially folded molten globule state is Klactalbumin ( for a review see [ 33 ]).
It is often useful to be able to quantify the contribution of multiple states to the unfolding of a protein.
There are several published algorithms that allow a set
of spectra which are obtained under different conditions to be deconvoluted into a smaller number of basis
curves. The original curves are a linear combination of
the basis curves. The amount of each basis curve contributing to each spectrum is then quantied. Two
widely used algorithms for deconvoluting sets of CD
spectra into basis curves are singular value decomposition (SVD ) ( reviewed in [ 34 ]) and the convex constraint algorithm ( CCA ) of Perczel et al. [ 35 ].
The CCA and SVD methods have been applied to
diverse problems in protein chemistry. For example,
Greeneld and Hitchcock-DeGregori [ 36 ] used CCA
to quantify the contribution of a molten globule-like
state to the unfolding of a designed coiled-coil peptide.
At 0C the peptide was 100% K-helical and had a
pronounced positive band at 280 nm arising from its
tyrosine residue. When heated from 0 to 50C, the
peptide lost all of its ellipticity in the near UV, while
retaining more than 75% of its K-helical content, suggesting that it went from a native to a molten-globule
state before completely dissociating to give disordered

chains. Deconvolution of the CD data obtained in the

far UV, as a function of temperature, showed that at
least three basis spectra were needed to t the data.
Safer et al. [ 28 ] similarly used the CCA algorithm
to examine the thermal stability and conformational
transitions of scrapie amyloid ( prion ) protein,
PrP27^30. CCA deconvolution of the CD spectra of
the solvent-exposed and rehydrated solid state PrP27^
30 identied ve common spectral components. The
loss of infectivity quantitatively correlated with a
decreasing proportion of native, L-sheet-like secondary structure component, an increasing amount
of an K-helical component, and an increasingly disordered tertiary structure. In a recent example, Konno
[ 37 ] has used SVD to analyze and quantify different
conformational states of acid denatured cytochrome
C.

Fig. 7. Kinetics of refolding ( A ) and unfolding ( B ) traces at

various nal guanidine-HCl concentrations and constant
peptide concentrations of the GCN4-P1 monitored by the
change in ellipticity at 222 nm at pH 7.0 and 5C. Solid
lines represent global ts to the model of a native dimer
dissociating to give two unfolded chains. Reprinted with
permission from [ 39 ]; z ( 1995 ) American Chemical Society.

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Fig. 8. Kinetic curves of the unfolding ( thin line ) and refolding ( thick line ) of RNAse HI as monitored by ( A ) far UV CD
due to the secondary structure at 220 nm, ( B ) near UV at
292 nm due to tertiary interactions, and ( C ) uorescence
( excitation at 280 nm and emission over 300 nm ). Only a
single exponential process was detected for unfolding
using all three probes. For refolding, the far UV CD value
largely recovered within 50 ms of the stopped-ow mixing
dead time ( burst phase ). This phase was followed by
either one phase detected by both near UV and far UV
CD with a rate constant of approximately 0.5^0.6 s31 .
Refolding detected by uorescence detected two phases,
one with a rate constant of 1.95 s31 and one with a rate
constant of 0.5 s31 corresponding to the phase detected
by near UV CD. Reprinted with permission from [ 40 ];
z ( 1995 ) American Chemical Society.

6. Kinetics of protein folding

Besides the study of protein folding under equilibrium conditions, CD can be used to measure rapid
events in protein folding and unfolding by attaching
a rapid mixing device to a CD spectrometer. Stoppedow CD has recently been reviewed by Kuwajima
[ 38 ]. Stopped-ow CD can be used both to obtain
the kinetic constants of folding reactions and to
dene the conformations of folding intermediates.
Data can be collected in the far and near UV.

In an elegant study of the development of secondary

structure as a function of time, Zitzewitz et al. [ 39 ]
have used stopped-ow CD to characterize the folding
of the GCN4-P1 peptide. This peptide is an K-helical
coiled-coil comprised of the leucine zipper domain of
the yeast GCN4 DNA transcription factor. The CD
data obtained during guanidine-HCl induced equilibrium unfolding of this peptide were consistent with a
two-state model involving the transition of a native Khelical coiled-coil dimer to an unfolded monomer. The
unfolding and refolding kinetics of GCN4-P1 were
monitored by stopped-ow CD a function of both peptide and denaturant concentration. The unfolding
kinetics displayed single-exponential behavior, consistent with a unimolecular reaction. The refolding
kinetics were dependent on both peptide and guanidine concentration and were well described by a simple bimolecular association reaction ( see Fig. 7 ). The
equilibrium constants calculated from the rate constants of folding and unfolding agreed with those
obtained from the equilibrium measurements, showing that all the data were consistent with a two-state
folding model.
Studies of changes in the near and far UV CD of
proteins, in parallel with other spectroscopic measurements, can give valuable information about the nature
of protein folding intermediates. In a recent example
illustrated in Fig. 8, Yamasaki et al. [ 40 ] used
stopped-ow CD and uorescence to study the refolding of ribonuclease HI from E. coli. Both the unfolding
and refolding processes, induced by concentration
jumps of either guanidine hydrochloride ( GuHCl ) or
urea, were studied. The peptide unfolds in a concerted
two-state transition. The difference in the amplitudes
of the burst phases for refolding, however, monitored
by uorescence and far and near UV CD spectra, suggest that an intermediate state exists with characteristics of a molten globule. Hydrogen exchange refolding competition combined with two-dimensional
NMR revealed that the amide protons of K-helix I
are the most highly protected, suggesting that Khelix I is the initial site of protein folding. The CD
and NMR data showed that the intermediate state
has a structure similar to that of the acid-denatured
molten globule.

7. Conclusion
Circular dichroism has many interesting and exciting uses for the study of protein and polypeptide structure, folding and interactions. The power of CD as an

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trends in analytical chemistry, vol. 18, no. 4, 1999

analytical tool is increased greatly when used in conjunction with other spectroscopic methods such as
uorescence and NMR. CD is an invaluable tool for
protein and peptide scientists, interested in understanding both the fundamental basis of macromolecular interactions and the nature of diseased states caused
by mutated or misfolded proteins.

Acknowledgements
I am grateful to Dr. Ernesto Freire for supplying an
original copy of the graph reproduced in Figure 4 and
I am indebted to Dr. Sarah E. Hitchcock-DeGregori
and Dr. Barbara Brodsky for critically reading the
manuscript. This work was supported by NIH Grants
GM36326 to S.E.H.D. and N.J.G. and HL35726 to
S.E.H.D. and by the CD facility at UMDNJ-Robert
Wood Johnson Medical School.

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