Applications of CD in Protein and Peptide Analysis

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trendsApplications of circular dichroism inprotein and peptide analysisNorma J. Green¢eldUMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA

This review discusses several useful applica-tions of circular dichroism as a tool for analyz-ing properties of proteins. The followingtopics are discussed: (1) protein^ligand inter-actions; (2) thermodynamics of protein fold-ing; (3) conformational transitions and pro-tein aggregation; (4) folding intermediates;(5) kinetics of protein folding. Speci¢c exam-ples are given to illustrate each applica-tion. z1999 Elsevier Science B.V. All rightsreserved.

Keywords: Circular dichroism; Protein-folding;Protein^ligand binding; Thermodynamics

1. Introduction

Circular dichroism (CD) is a valuable spectro-scopic technique for studying protein structure in sol-ution because many common conformational motifs,including K-helixes, L-pleated sheets, poly-L-prolineII-like helices and turns, have characteristic far UV(178^250 nm) CD spectra. For example, K-helicesdisplay large CD bands with negative ellipticity at222 and 208 nm, and positive ellipticity at 193 nm,L-sheets exhibit a broad negative band near 218 nmand a large positive band near 195 nm, while disor-dered extended chains have a weak broad positive CDband near 217 nm and a large negative band near 200nm. The spectrum of a protein is basically the sum ofthe spectra of its conformational elements, and thusCD can be used to estimate secondary structure. Inaddition, the chromophores of the aromatic aminoacid proteins are often in very dissymmetric environ-ments resulting in distinctive CD spectra in the nearUV (250^300 nm), which can serve as useful probesof protein tertiary structure. Recently several articles[ 1^3 ] have compared and evaluated most of the cur-rently 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 facetsthan simply being a tool to estimate protein structure.For example, it is an excellent technique for determin-ing the thermodynamics and kinetics of protein fold-ing and denaturation and is unsurpassed for followingthe effects of mutations on protein folding and stabil-ity. In addition, it can be an excellent tool for followingprotein^ligand interactions. As sophisticated mathe-matical programs for ¢tting nonlinear equations anddeconvoluting sets of curves using personal com-puters have become increasingly available, the analy-sis of thermodynamic and kinetic experiments, whichcan be performed by following changes in CD spectra,have become much simpler, increasing the usefulnessof the technique.

It would be impossible to do a complete review ofall of the recent applications involving CD spectros-copy. For example, in the last 10 years there have beenmore than 1500 papers published in the Journal ofBiological Chemistry alone, where CD was used tostudy various aspects of protein and nucleic acidchemistry. This review will therefore focus on severalspeci¢c applications that illustrate of the general use-fulness of CD as an analytical tool. The followingtopics will be discussed: (1 ) protein^ligand interac-tions; (2 ) thermodynamics of protein folding; (3 )conformational transitions and protein aggregationphenomena; (4 ) folding intermediates; and (5)kinetics of protein folding. Speci¢c examples will beused to illustrate each application.

2. Protein^ligand interactions

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

0165-9936/99/$ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 - 9 9 3 6 ( 9 8 ) 0 0 1 1 2 - 5

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

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the CD spectrum of each component in a solution isdirectly proportional to its concentration, and the totalspectrum represents the sum of all the contributingspectra. Thus, the change in the CD spectrum of aprotein upon addition of a ligand, denaturant or heatis directly proportional to the amount of proteinchanged by the perturbation.

Circular dichroism has been used to study protein^ligand interactions since the instrumentation ¢rstbecame available in the late 1960s [ 4 ]. Circulardichroism can be used to follow the binding of ligandsto proteins, peptides and nucleic acids provided one oftwo criteria is ful¢lled. Either the binding of ligandmust bind in an dissymmetric fashion that inducesextrinsic optical activity in the chromophores of thebound ligand, or the binding must result in a confor-mational change in the macromolecule that results in achange in its intrinsic CD spectrum. Before ef¢cientalgorithms for solving nonlinear equations becamewidely available, it was usually necessary to linearizethe binding equations to obtain binding constants fromCD data ( reviewed in [ 5 ]). Today it is usual to ¢t thenonlinear binding equations directly using commer-cial programs such as SigmaPlot0, Origin0, Graph-Pad0 and PsiPlot0, among others, which use ¢ttingtechniques such as the Levenberg^Marquardt algo-rithm [ 6 ] to estimate the constants which best ¢t thedata.

2.1. Extrinsic effects

When highly chromatic ligands bind to proteinsthey may generate large CD bands, called Cottoneffects, in the region of their absorbance spectrumif they bind in a dissymmetric fashion. The changein ellipticity as a function of substrate concentra-tion 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 theconcentration of protein^ligand complex formed.The equilibrium constant, K, obeys the followingequation:

K � ��LPi��=��Li�3n�LPi��Po�3�LPi�� 1�

The change in ellipticity, ai, due to complex formationis directly proportional to [LPi ]. When all of the pro-tein has bound ligand, ai = amax

�LPi� � ��Po��ai=amax� �2�

ai � amaxf�1� K�Po�=n� K�Po��=2K�Po�3f��1� K�Li�=n� K�Po��=2K�Po��2

3��Li�=n�Po��g1=2g �3�

By ¢tting Eq. 3 to the raw data, the binding constantof the ligand for the protein and the number of bindingsites can be estimated. To illustrate, when folates andnucleotides bind to dihydrofolate reductase large CDbands are induced [ 7 ]. Fig. 1A shows the extrinsicCotton effects induced in the near UV when dihydro-folate, folate and methotrexate bind to dihydrofolatereductase from Escherichia coli, and Fig. 1B showsthe change in ellipticity as a function of added dihy-

Fig. 1. (A) The molar circular dichroism of dihydrofolatereductase and 1:1 complexes with dihydrofolate (dashedline), folate (dot-dashed line) and methotrexate (solidline). (B) The increase in ellipticity at 292.5 nm as a func-tion of dihyrofolate concentration: (solid line) enzyme plusdihydrofolate, (dashed line) dihydrofolate alone. Re-printed with permission from [ 7 ]; z (1972) AmericanChemical Society.

trends in analytical chemistry, vol. 18, no. 4, 1999 237

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drofolate. The data were used to determine a bindingconstant of 1.1 WM.

As well as being used to study the interactions ofsubstrates and inhibitors with enzymes, extrinsic Cot-ton effects have been used to study protein^ligandinteractions in diverse systems. For example, theyhave been used to study the binding of cofactors toproteins including the binding of nucleotides to lacticdehydrogenase [ 8 ], ATP to GroEL [ 9 ], pyridoxalphosphate to tryptophan synthase [ 10 ] and the bind-ing of retinol to interphotoreceptor retinol bindingprotein [ 11]. In addition extrinsic CD bands havebeen employed to study the interactions of drugswith bovine serum albumin (e.g. [ 12 ]) and the inter-actions of DNA binding proteins with DNA (e.g.[ 13 ]).

2.2. Intrinsic effects

The binding of ligands to proteins often results inconformational transitions. The resultant change in theintrinsic ellipticity of the backbone amides caused bythe conformational change can also be used to obtainthe ligand binding constants. For example, divalentcations are effectors in a many biological systemsthat often induce large conformational changes in tar-get proteins. For example, Ca2� ion binding to themuscle regulatory protein, troponin C, induces anincrease in ellipticity due to a stabilization ofK-helicalregions of the protein. Johnson and Potter [ 14 ]showed that CD could distinguish two classes ofCa2� binding sites in troponin C, with high and lowaf¢nity. Calcium binding to the low af¢nity site regu-lates muscle contraction. Fig. 2 illustrates how thechange in ellipticity of troponin C (TnC) as a functionof free calcium concentration can be used to obtain thecalcium binding constants for the high and low af¢nitysites, ka1 and ka2 [ 15 ]. Data obtained with skeletalmuscle TnC, TnC expressed in E. coli, and a mutantTnC lacking a helical segment at the N-terminus ( theN-helix ) are illustrated. Chelators that bind Ca2� wereused as buffering agents so that the free Ca2� concen-tration was ¢xed at the desired concentrations. Thechanges in ellipticity at 222 nm as a function of freeCa2� concentration were normalized to a scale of 0 (noCa2� ) to 1 (saturating Ca2� ) and ¢t to the sum of twoHill equations:

ai � �a1kH1a1 �Ci�H1�=�1� kH1

a1 �Ci�H1��a2kH2

a2 �Ci�H2�=�1� kH2a2 �Ci�H2� �4�

where ai is the change in ellipticity at 222 nm observedwhen calcium binds to TnC, H1 and H2 are the Hillconstants and a1 and a2 are the molar absorptivities forthe spectral change caused by calcium binding to thehigh and low af¢nity sites, respectively, and [Ci ] is thefree 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 decreasein the binding of calcium to the regulatory site of TnC[ 15 ].

There are many other classes of protein^ligandinteractions that result in conformational changes.For example, DNA binding to DNA transcription fac-tors results in an increase in helical content of the tran-scription factor. These factors often contain K-helicalcoiled-coils or helix-loop-helix motifs, which serve asdimerization domains, and a relatively unstructuredbasic region which binds to DNA. Upon binding tothe DNA the basic region assumes a helical conforma-tion. Fig. 3 illustrates the increase in ellipticity whentwo muscle helix-loop-helix regulatory factors, E47and MyoD bind to an oligonucleotide containing thecreatine enhancer site [ 16 ].

Another interesting class of protein^ligand interac-tions, resulting in conformational changes, is the bind-

Fig. 2. The increase in ellipticity of troponin C (TnC) at 222nn, due to increasedK-helical content, as a function of freeCa2� concentration. R, muscle TnC; a, wild type TnCexpressed in E. coli; b, recombinant TnC missing the¢rst 14 N-terminal residues. Ellipticities at 222 nm are nor-malized to a scale of 0 (no Ca2� ) to 1 (saturating Ca2� ).The points are the raw data, and the lines are the data ¢ttedby the sum of two Hill equations. Reprinted with permis-sion from [ 15 ]; z (1994) The American Society for Bio-chemistry and Molecular Biology, Inc.


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ing of heparin to diverse proteins including the amino-terminal domain of ¢bronectin [ 17 ], hepatocytegrowth factor [ 18 ], the amyloid precursor protein ofAlzheimer's disease [ 19 ] and annexin [ 20 ] amongothers. The binding of heparin has very differenteffects, depending on its target. CD studies showthat a peptide derived from the amyloid precursor pro-tein, APP416^447 shifts towards an K-helical confor-mation in the presence of heparin. In contrast, theCa2�-dependent binding of heparin to annexin causeda large decrease in its K-helical content from approx-imately 44 to 31%, a small decrease in the L-sheetcontent from approximately 27 to 24%, and anincrease in the unordered structure from 20 to 29%.A peptide fragment corresponding to the heparin bind-ing site of hepatocyte growth factor changes from arandom structure to a L-sheet-like structure upon hep-arin binding, which results in the oligomerization ofthe growth factor.

3. Thermodynamics of protein folding

Following changes in the optical activity of a pro-tein or polypeptide as a function of temperature ordenaturant is a very convenient method of obtainingthe thermodynamics of folding. Indeed, the majorapplication of CD today is the determination of thethermodynamics of protein folding and the effects ofmutations on protein stability. The equations describ-ing the thermodynamics of structural stability andcooperative folding behavior in proteins have beenreviewed by Freire [ 21]. The applications of CD tostudies of protein folding have been recently reviewedby Kelly and Price [ 22 ].

3.1. Thermodynamic parameters of folding fromthermal denaturation and renaturation studies

Essentially the use of CD to follow protein denatu-ration depends on the fact that the change in ellipticityis directly proportional to the change in concentrationof native and denatured forms. The observed elliptic-ity of a protein, aobs, changes when it unfolds. Whenthe protein is fully folded aobs = a1 and when it is fullyunfolded aobs = a2. In the case of a monomeric protein,the equilibrium constant of folding, K = folded /unfolded. If we de¢ne K as the fraction folded at agiven temperature then

K � K=�13K� �5�

vG � nRTlnK �6�

where vG is the free energy of folding and R is the gasconstant = 1.987 cal / mol.

vG � vH3TvS �7�

where vH is the enthalpy of folding and vS is theentropy of folding. TM is the observed midpoint ofthe thermal transition. At the TM, K = 1, thereforevG = 0 and vS =vH /TM. Solving these equations weobtain:

K � expf�vH=RT��T=TM�31�g �8�

K � K=�1� K� �9�

aobs � �a13a2�K� a2 �10�

To calculate the values of vH and TM that bestdescribe the folding curve, initial values of vH, TM,

Fig. 3. The backbone ellipticity of two muscle DNA tran-scription factors: a, E47 and E, MyoD, and O, a chimera,MyoEL, in which the loop region of MyoD has beenreplaced by that of E47. The CD spectra of all three pro-teins are similar and are characteristic of a partially helicalstructure. Addition of an oligonucleotide containing thecreatine kinase enhancer site to b, E47 or F, MyoDincreases negative ellipticity at 222 and 208 nm character-istic of K-helices, because the basic region folds uponbinding to DNA. Reprinted with permission from [ 16 ];z (1998) The American Society for Biochemistry andMolecular Biology, Inc.


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a1 and a2 are estimated, and Eq. 10 is ¢tted to theexperimentally observed values of the change in ellip-ticity as a function of temperature, by a curve ¢ttingroutine such as the Levenberg^Marquardt algorithm[ 6 ]. Since at K = 1, vG = 0, the entropy of folding cantherefore be calculated using Eq. 4 where vS =vH /TM. The free energy of folding at any other temper-ature can then be calculated using Eq. 4. Note that thistreatment ignores changes in heat capacity, vCp, whena protein unfolds, but equations have been developedthat include that term [ 23 ]. Similar equations can beused to estimate the thermodynamics of folding ofproteins and peptides that undergo folded multimerto unfolded monomer transitions ( reviewed in [ 24 ]).The free energy of folding may also be calculatingfrom the concentration dependence of CD spectra, ifchanges in association state are accompanied bychanges in conformation.

Fig. 4 illustrates how changes in ellipticity as afunction of temperature and concentration have beenused to determine the enthalpy of folding of the GCN4transcription factor, which undergoes a two-state tran-sition between a folded two-stranded K-helical coiled-coil and a monomeric disordered state [ 25 ]. The val-ues for the enthalpy of the folding transition, deter-mined from the changes in ellipticity as a function oftemperature agreed with the values obtained fromscanning calorimetry.

3.2. Thermodynamic parameters of proteinfolding using denaturants

In some cases thermal folding and unfolding studiesare impractical. The protein may have a very high TM,or may aggregate and precipitate upon heating. Inthese cases denaturants, such as urea or guanidine-HCl, may be added to a protein or peptide to induceunfolding. One obtains K at each concentration ofdenaturant by assuming that the protein is native inthe absence of denaturant, and fully unfolded whenthere is no further change in ellipticity upon additionof higher concentrations of perturbant. In each case, K,the equilibrium constant of folding is determined from

Fig. 5. (A) Concentration dependence of the guanidinehydrochloride 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 con-centration of guanidine hydrochloride at different concen-trations of the peptide, extrapolated to zero to determinethe free energy of unfolding in the absence of denaturant.The correlation coef¢cient for the regression line was 0.94.Reprinted with permission from [ 26 ];z (1992) The Amer-ican Society for Biochemistry and Molecular Biology, Inc.

Fig. 4. The change in fraction folded of GCN4 as a functionof temperature and peptide concentration. The concentra-tions range from 1 WM with the lowest TM to 20 WM withthe highest TM. The enthalpy of unfolding was 35.0 þ 1.1kcal / mol ( monomer) which compared to that of 34.7 þ 0.3kcal / mol measured by calorimetry. Reprinted with per-mission from [ 25 ]; z (1993) American Chemical So-ciety.


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the ellipticity observed at each concentration of denat-urant.

K � K=�n�C�n31��13K�n �11�where

K � �aobs3a2�=�a13a2� �12�

K, a1 and a2 have the same de¢nitions as in the case ofthermal unfolding above. [ C ] is the total concentra-tion of protein monomers. The free energy of folding isevaluated at every concentration of denaturant usingthe equation vG =3nRTlnK. The free energies areplotted as a function of denaturant and extrapolatedto zero denaturant to obtained the free energy of fold-ing of the native material. Hodges and coworkers haveused this approach to study the contributions of hydro-phobic and charged residues to the stability ofdesigned coiled-coil K-helices. Fig. 5 illustrates thedetermination of the free energy of folding of acoiled-coil model peptide from a guanidine denatura-tion study [ 26 ]. It should be noted that one may obtaindifferent apparentvGs of folding when different dena-turants are employed because some denaturants, suchas solutions of guanidine-HCl, which have a high ionicstrength, disrupt salt bridges in proteins, while others,such as urea, which is uncharged, have a lesser effecton protein charge^charge interactions.

4. Conformational transitions

While CD is most usually used to study transitionsfrom a folded to an unfolded state, recently it has beenfound that certain systems undergo transitions involv-ing changes from an K-helical to a L-pleated sheetconformation (or vice versa ). Conformational transi-tions may be followed by oligomerization of the pro-tein and /or aggregation and precipitation. Such con-formational changes can have important clinicalconsequences as it is felt that such transitions maybe involved in the pathology of diseases such as scra-pie, `mad cow disease' and Alzheimer's disease. CDhas been used to follow conformational transitions inprions (e.g. [ 27 ]), the Alzheimer's L-amyloid peptide(e.g. [ 28 ]) and amylin, an amyloid peptide seen in thepancreatic islets in patients with diabetes (e.g. [ 29 ]).Fig. 6 illustrates conformational changes of islet amy-loid polypeptide after seeding with peptide modi¢edwith advanced glycosylation end products [ 30 ]. Thepeptide rapidly goes from a conformation that is pre-dominantly random, with a small amount ofK-helix, toa conformation with considerable L-structure. Afterthe structural transition, the peptide associates into¢brils and precipitates out of solution.

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


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5. Folding intermediates

Often when proteins or peptides fold or unfold, theprocess is not a two-state transition between the nativeand totally disordered forms; there are intermediatestates. These states may be transient, and may beobserved during kinetic measurements of folding orunfolding, or they may be stable intermediates, seenwhen the peptide is subject to denaturing conditions,such as high temperatures, or exposure to urea, guani-dine or detergents. These states may be partiallyfolded, with de¢ned tertiary structures, or may be socalled molten globules. The term molten globule was¢rst introduced by Ptitsyn and coworkers ( for reviewssee [ 31,32 ]) to describe a compact state with native-like secondary structure but slowly £uctuating tertiarystructure.

CD can often be used to detect and quantify theintermediate states. For example, some `molten glob-ule' folding intermediates exhibit large CD signals inthe far UV arising from secondary structural elementssuch as K-helices. The molten globule folding inter-mediates, however, display almost none of the nearUV CD bands arising from tertiary structural interac-tions of aromatic groups, which are often seen in thespectra of native proteins. A classic example of a pro-tein with a partially folded molten globule state is K-lactalbumin ( for a review see [ 33 ]).

It is often useful to be able to quantify the contribu-tion of multiple states to the unfolding of a protein.There are several published algorithms that allow a setof spectra which are obtained under different condi-tions to be deconvoluted into a smaller number of basiscurves. The original curves are a linear combination ofthe basis curves. The amount of each basis curve con-tributing to each spectrum is then quanti¢ed. Twowidely used algorithms for deconvoluting sets of CDspectra into basis curves are singular value decompo-sition (SVD) ( reviewed in [ 34 ]) and the convex con-straint algorithm (CCA) of Perczel et al. [ 35 ].

The CCA and SVD methods have been applied todiverse problems in protein chemistry. For example,Green¢eld and Hitchcock-DeGregori [ 36 ] used CCAto quantify the contribution of a molten globule-likestate to the unfolding of a designed coiled-coil peptide.At 0³C the peptide was 100% K-helical and had apronounced positive band at 280 nm arising from itstyrosine residue. When heated from 0 to 50³C, thepeptide lost all of its ellipticity in the near UV, whileretaining more than 75% of its K-helical content, sug-gesting that it went from a native to a molten-globulestate before completely dissociating to give disordered

chains. Deconvolution of the CD data obtained in thefar UV, as a function of temperature, showed that atleast three basis spectra were needed to ¢t the data.Safer et al. [ 28 ] similarly used the CCA algorithmto examine the thermal stability and conformationaltransitions of scrapie amyloid (prion) protein,PrP27^30. CCA deconvolution of the CD spectra ofthe solvent-exposed and rehydrated solid state PrP27^30 identi¢ed ¢ve common spectral components. Theloss of infectivity quantitatively correlated with adecreasing proportion of native, L-sheet-like sec-ondary structure component, an increasing amountof an K-helical component, and an increasingly disor-dered tertiary structure. In a recent example, Konno[ 37 ] has used SVD to analyze and quantify differentconformational states of acid denatured cytochromeC.

Fig. 7. Kinetics of refolding (A) and unfolding (B) traces atvarious ¢nal guanidine-HCl concentrations and constantpeptide concentrations of the GCN4-P1 monitored by thechange in ellipticity at 222 nm at pH 7.0 and 5³C. Solidlines represent global ¢ts to the model of a native dimerdissociating to give two unfolded chains. Reprinted withpermission from [ 39 ]; z (1995) American Chemical Soci-ety.


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6. Kinetics of protein folding

Besides the study of protein folding under equilib-rium conditions, CD can be used to measure rapidevents in protein folding and unfolding by attachinga rapid mixing device to a CD spectrometer. Stopped-£ow CD has recently been reviewed by Kuwajima[ 38 ]. Stopped-£ow CD can be used both to obtainthe kinetic constants of folding reactions and tode¢ne the conformations of folding intermediates.Data can be collected in the far and near UV.

In an elegant study of the development of secondarystructure as a function of time, Zitzewitz et al. [ 39 ]have used stopped-£ow CD to characterize the foldingof the GCN4-P1 peptide. This peptide is an K-helicalcoiled-coil comprised of the leucine zipper domain ofthe yeast GCN4 DNA transcription factor. The CDdata obtained during guanidine-HCl induced equilib-rium unfolding of this peptide were consistent with atwo-state model involving the transition of a native K-helical coiled-coil dimer to an unfolded monomer. Theunfolding and refolding kinetics of GCN4-P1 weremonitored by stopped-£ow CD a function of both pep-tide and denaturant concentration. The unfoldingkinetics displayed single-exponential behavior, con-sistent with a unimolecular reaction. The refoldingkinetics were dependent on both peptide and guani-dine concentration and were well described by a sim-ple bimolecular association reaction (see Fig. 7 ). Theequilibrium constants calculated from the rate con-stants of folding and unfolding agreed with thoseobtained from the equilibrium measurements, show-ing that all the data were consistent with a two-statefolding model.

Studies of changes in the near and far UV CD ofproteins, in parallel with other spectroscopic measure-ments, can give valuable information about the natureof protein folding intermediates. In a recent exampleillustrated in Fig. 8, Yamasaki et al. [ 40 ] usedstopped-£ow CD and £uorescence to study the refold-ing of ribonuclease HI from E. coli. Both the unfoldingand refolding processes, induced by concentrationjumps of either guanidine hydrochloride (GuHCl) orurea, were studied. The peptide unfolds in a concertedtwo-state transition. The difference in the amplitudesof the burst phases for refolding, however, monitoredby £uorescence and far and near UV CD spectra, sug-gest that an intermediate state exists with character-istics of a molten globule. Hydrogen exchange refold-ing competition combined with two-dimensionalNMR revealed that the amide protons of K-helix Iare the most highly protected, suggesting that K-helix I is the initial site of protein folding. The CDand NMR data showed that the intermediate statehas a structure similar to that of the acid-denaturedmolten globule.

7. Conclusion

Circular dichroism has many interesting and excit-ing uses for the study of protein and polypeptide struc-ture, folding and interactions. The power of CD as an

Fig. 8. Kinetic curves of the unfolding ( thin line) and refold-ing ( thick line) of RNAse HI as monitored by (A) far UV CDdue to the secondary structure at 220 nm, (B) near UV at292 nm due to tertiary interactions, and (C) £uorescence(excitation at 280 nm and emission over 300 nm). Only asingle exponential process was detected for unfoldingusing all three probes. For refolding, the far UV CD valuelargely recovered within 50 ms of the stopped-£ow mixingdead time ( burst phase). This phase was followed byeither one phase detected by both near UV and far UVCD 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 rateconstant of 0.5 s31 corresponding to the phase detectedby near UV CD. Reprinted with permission from [ 40 ];z (1995) American Chemical Society.


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analytical tool is increased greatly when used in con-junction with other spectroscopic methods such as£uorescence and NMR. CD is an invaluable tool forprotein and peptide scientists, interested in under-standing both the fundamental basis of macromolecu-lar interactions and the nature of diseased states causedby mutated or misfolded proteins.

Acknowledgements

I am grateful to Dr. Ernesto Freire for supplying anoriginal copy of the graph reproduced in Figure 4 andI am indebted to Dr. Sarah E. Hitchcock-DeGregoriand Dr. Barbara Brodsky for critically reading themanuscript. This work was supported by NIH GrantsGM36326 to S.E.H.D. and N.J.G. and HL35726 toS.E.H.D. and by the CD facility at UMDNJ-RobertWood Johnson Medical School.

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Applications of CD in Protein and Peptide Analysis

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