REVIEW / SYNTHESE

Mutational investigation of protein foldingtransition states by F-value analysis and beyond:lessons from SH3 domain folding1

Arash Zarrine-Afsar, Sung Lun Lin, and Philipp Neudecker

Abstract: Understanding how proteins adopt their unique native structures requires a complete structural characterizationof the rate-limiting transition state(s) along the folding pathway. By definition, transition states are not significantly popu-lated and are only accessible via folding kinetics studies. In this respect, interpreting the kinetic effects of amino acid sub-stitutions (especially to Ala) via F-value analysis is the most common method to probe the structure of these transient, yetimportant states. A critical review of the key assumptions required for rigorous interpretation of F values reveals that amultiple substitution strategy in which a position of interest is mutated to a variety of amino acids, and not exclusively toAla, provides the best means to characterize folding transition states. This approach has proven useful in revealing non-native interactions and (or) conformations in folding transition states. Moreover, by simultaneously examining the foldingkinetics of multiple substitutions made at a single surface-exposed position using the Brønsted analysis the backbone con-formation in a folding transition state can be investigated. For folding equilibria with exchange rates on the order of milli-seconds, the kinetic parameters for F-value analysis can be obtained from NMR relaxation dispersion experiments, underfully native conditions, along with a wealth of high-resolution structural information about the states in exchange (native,denatured, and intermediate states that populate the pathway). This additional structural information, which is not readilyobtained through stopped-flow based methods, can significantly facilitate the interpretation of F values because it often re-ports on the validity of the assumptions required for a rigorous interpretation of F values.

Key words: protein folding, F-value analysis, SH3 domains, mutagenesis, folding transition states.

Resume : La comprehension du mecanisme par lequel les proteines adoptent leurs structures natives uniques requiert unecaracterisation structurale complete de l’etat ou des etats de transitions limitants durant le processus de repliement. Par de-finition, les etats de transition ne sont pas significativement abondants et ne sont accessibles que par l’intermediaired’etudes cinetiques de repliement. A cet effet, l’interpretation des effets cinetiques de la substitution d’acides amines (spe-cialement par une Ala) a l’aide d’une analyse des valeurs de F est la methode la plus utilisee pour sonder ces etats transi-toires mais importants. Une revue critique des postulats de base requis pour interpreter rigoureusement les valeurs de F

revele qu’une strategie utilisant de multiples substitutions, par laquelle un acide amine situe dans une position d’interet estmute en une variete d’acides amines et non seulement en Ala, fournit le meilleur moyen de caracteriser les etats de transi-tion de repliement. Cette approche a demontre son utilite en revelant les interactions/conformations non-natives dans lesetats de transition de repliement. De plus, en examinant simultanement des cinetiques de repliement de multiples substitu-tions realisees sur un site unique expose a la surface par une analyse de Brønsted, la conformation d’une chaıne dans unetat de transition de repliement donne peut etre etudiee. Dans le contexte d’un repliement a l’equilibre avec des tauxd’echange de l’ordre de la milliseconde, les parametres cinetiques de l’analyse des valeurs de F peuvent etre obtenus pardes experiences en RMN de relaxation/dispersion, sous des conditions pleinement natives, avec une abondance d’informa-tions structurales a haute resolution des etats en echange (natif, denature et etats intermediaires qui jalonnent ce sentier).Cette information structurale additionnelle, qui n’est pas facilement obtenue par des methodes en stop-flow, peut faciliter

Received 21 July 2009. Revision received 10 September 2009. Accepted 25 September 2009. Published on the NRC Research Press Website at bcb.nrc.ca on 12 March 2010.

A. Zarrine-Afsar.2 Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Chemistry,University of Toronto, Toronto, ON M5S 3H6, Canada.S.L. Lin. Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.P. Neudecker. Department of Chemistry, Department of Biochemistry, and Department of Molecular Genetics, University of Toronto,Toronto, ON M5S 1A8, Canada.

1This paper is one of a selection of papers published in this special issue entitled ‘‘Canadian Society of Biochemistry, Molecular &Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases’’ and has undergone the Journal’s usual peer reviewprocess.

2Corresponding author (e-mail: arash.zarrine.afsar@utoronto.ca).

231

Biochem. Cell Biol. 88: 231–238 (2010) doi:10.1139/ O09-153 Published by NRC Research Press

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de facon significative l’interpretation des valeurs de F car elle temoigne de la validite des postulats requis pour interpreterrigoureusement les valeurs de F.

Mots-cles : repliement des proteines, analyse des valeurs de F, domaines SH3, mutagenese, etats de transition de replie-ment.

[Traduit par la Redaction]

Introduction to F-value analysisIn the quest to understand the biophysical principles

underlying the relationship between the amino acid sequenceof a protein and its three-dimensional structure, many stud-ies have used proteins that exhibit a two-state (U$F) fold-ing mechanism in which the attainment of the folded state(F), in equilibrium with the unfolded state (U), is precededby a high-energy, transiently populated barrier, referred toas the transition state ({). In particular, kinetics studies in-vestigating the structure of protein-folding transition stateshave been instrumental in advancing our understanding ofthe key interactions that drive folding. Introduced by thepioneering work of the Fersht laboratory (Matouschek et al.1989), F-value analysis is the most commonly used methodto probe the transition state structures in protein folding. Theobjective of this technique is to evaluate the importance of amutated (generally to Ala) residue in stabilizing the foldingtransition state structure. By performing F-value analysis(Fig. 1) on mutations made at multiple positions within aprotein, it becomes possible to map out which regions ofthe protein molecule are structured in the transition state.Through comparing the experimentally determined folding(kf) and unfolding (ku) rates of mutant (mut) and wild type(wt) proteins, the changes in the stability of the folding tran-sition state (DDG{?u) and the equilibrium free energy ofunfolding (DDGf?u) are calculated to obtain a F-value asfollows:

F = DDG{?u/DDGf?u

where DDGf?{ = –RTln(kuwt/ku

mut, DDG{?u = –RTln(kfmut/

kfwt, and DDGf?u = DDGf?{ + DDG{?u.A F value of unity indicates that the mutated side chain

is involved in energetically similar interactions in both thefolded and transition state, and that the native structure sur-rounding this side chain is formed in the transition state. Onthe other hand, a F value of 0 results from side chains thatadopt their native structures after the formation of the transi-tion state. For these residues, the native interactions are aspoorly formed in the transition state as they are in the un-folded state. In practice, however, many reported F valuesrange between 0 and unity, and these can only be interpretedrigorously under certain conditions (Fersht and Sato 2004).To obtain meaningful F values the following critical as-sumptions must be made:

1. Ala mutagenesis must only result in a net deletion of aninteraction, and no new interactions in a folding transi-tion state should be introduced by the mutation.

2. The wild type and mutant protein must follow the samefolding pathway. Hence, the mutants used should not bedisruptive of the folding pathway of the wild type pro-tein.

3. The folding transition state is assumed to be exclusivelystabilized by native-like interactions (without formingany non-native interactions).

4. The structure of the unfolded state (commonly used asthe reference state) should not be altered upon mutagen-esis, and the unfolded state is believed to be devoid ofsignificant residual structure.

5. Mutants used should not be disruptive of the folded statestructure, although there is little crystallographic evi-dence that point mutations in T4 lysozyme (Xu et al.1998), and in a number of other systems investigated ,can dramatically change the folded state structure.

6. Mutations should not drastically change the solvation en-ergy (i.e., where a polar residue is substituted with anon-polar side chain or vice a versa), because an altera-tion of the solvation energy may lead to an artifactual Fvalue (Fersht and Sato 2004).

Therefore, for the purpose of F-value analysis, ‘‘conser-vative’’ substitutions that are non-disruptive of both thestructure and the folding pathway of the wild type proteinmust be used. An ideal conservative substitution is Ile toVal, which only eliminates one methylene group, withoutcausing any other disruptions in the structure. In practice,however, very conservative substitutions often fail to pro-

Folding coordinate

Fre

e e

ne

rgy

U

F

‡wtmut ΔΔG‡→u

ΔΔGf→u

Fig. 1. A typical energy diagram for F-value analysis on a proteinfollowing the two-state folding mechanism. The F values can becalculated from the depicted energy differences according to eq. 1.The energy differences shown can be calculated from the kineticrate constants of folding and unfolding of the protein, as describedin the text.

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duce large enough changes in the DDGf?u to the level nec-essary for obtaining reliable F values, and most protein-en-gineering studies have used substitutions with Ala almostexclusively regardless of the type of residue being substi-tuted.

The advantages of performing F-valueanalysis on surface-exposed positions

The majority of amino acid side chains in a protein struc-ture are simultaneously involved in multiple interactions.Consequently, substitutions made in the hydrophobic corepositions often cause large changes in the overall stability(DDGf?u) of the protein. As a consequence of the tight vander Waals packing in the hydrophobic core, some of the mu-tants used to probe the F values of core positions might betoo destabilizing to fold on their own, or violate assumption5 above. Moreover, there is evidence that van der Waalspacking interactions in the hydrophobic core transition statemay be drastically different from those seen in the foldedstate (Northey et al. 2002a), and this may cause complica-tions in the rigorous interpretation of F values for the hy-drophobic core positions. For example, Ala mutagenesis ofa core position that is loosely packed in the transition state,but tightly packed in the folded state, may result in a F

value that is fairly low in magnitude—for such a mutation,the DDG{?u is, by definition, going to be smaller thanDDGf?u. In addition, mutagenesis of a residue that is non-optimally packed in the core of the folded state, but is ener-getically favorable in the transition state (due to its looserpacking requirement), may lead to a F value of greaterthan unity (F > 1), which is difficult to interpret within theframework of classical F-value analysis proposed by theFersht laboratory (Ala mutagenesis at this position willcause a greater destabilization in the transition state(DDG{?u) compared with the folded state (DDGf?u)(Northey et al. 2002a), leading to a non-canonical F value).

On the other hand, at surface-exposed positions, where aside chain is involved in minimal tertiary contacts, the ob-served energetics are not confounded by packing interac-tions, and are more easily interpreted. However, sincesurface-exposed residues possess equal solvent exposures inthe folded and unfolded states, their contribution to proteinstability is often thought of as insignificant, and mostprotein-engineering studies to enhance the thermodynamicstability have exclusively focused on optimizing packing in-teractions in the hydrophobic core (Dahiyat and Mayo1997). Quite contrary to this common perception, manystudies suggest that mutagenesis of surface-exposed posi-tions can have a significant effect on protein stability, assurface-exposed residues are often involved in local struc-ture stabilization (local structure propensity effects) (Kimand Berg 1993; Minor and Kim 1994b; Zarrine-Afsar et al.2007), or long-range electrostatics (Sanchez-Ruiz and Ma-khatadze 2001; Permyakov et al. 2005; Schweiker et al.2007). For example, the optimization of surface-exposedlong-range electrostatic interactions in the Fyn SH3 domain(four negative to positive charge reversal mutations and oneintroduction of a favorable positive charge to eliminate un-favorable charge–charge interactions) stabilized this proteinby ~1.8 kcal�mol–1, a 40% increase in stability (Schweiker

et al. 2007). Likewise, the energetic contribution of localstructure propensity at exposed positions has been shown tobe significant, with DDGf?u values of ~2.0 kcal�mol–1 ob-served for surface-exposed b-strand positions mutated inthe Fyn SH3 domain (Zarrine-Afsar et al. 2007). Surface-exposed positions are well suited for experimentally inves-tigating the energetics of local structure propensity withfewer confounding effects of tertiary interactions, and exper-imental verification of database-derived propensities willfurther validate the use of these propensity scales in struc-ture prediction algorithms.

F-value analysis taken one step further:using multiple amino acid substitutions toprobe folding transition states

As discussed previously, the suitability of non-conservativesubstitutions for the purpose of analyzing protein foldingtransition states has been debated. Surprisingly, in the stud-ies examining the effect on folding kinetics of a widevariety of substitutions at single positions in the Fyn SH3domain (Northey et al. 2002a, 2002b; Di Nardo et al. 2004)similar F values were obtained independent of the nature ofthe residue being substituted. Therefore, the folding transi-tion state of the Fyn SH3 domain was deemed tolerant ofmultiple substitutions. Consistent with this notion, the Srcand Spc SH3 domains, which display only 33% sequenceidentity, have been shown to possess very similar transition-state structures (Martınez Serrano 1999). To examine thegenerality of this observation, direct F-value comparisonsfor aligned positions in 7 pairs of structurally similar pro-teins (RMSD of 0.81 to 3.03 A) with amino acid identitiesranging from 75% to 4% were made (Zarrine-Afsar et al.2005). Remarkably, good overall quantitative agreement be-tween the F values of structurally equivalent positions inthese different systems was seen (Zarrine-Afsar et al. 2005),suggesting that proteins with similar structures and divergentsequences do share common folding transition-state struc-tures. Therefore, it appears that so long as the substitutionsmade do not drastically alter the topology of the wild typeprotein, the mutant variants of the model systems examinedearlier are likely to follow the same folding pathway as thewild type protein. This observation may be relevant for themutational analysis of folding transition states of non-SH3domain systems, and the power of the multiple substitutionsstrategy promises a deeper understanding of the forces thatstabilize these transient, yet important, states. By correlatingthe experimental folding kinetic data of each mutant withappropriate pseudo-energy terms derived from comparingthe physicochemical properties of the mutant and the wildtype side chains, this approach has proven useful in dissect-ing the folding energetics of local structure propensity(Northey et al. 2002b; Zarrine-Afsar et al. 2007), hydro-phobicity (Zarrine-Afsar et al. 2008), and packing (Northeyet al. 2002a). For example, by comparing the folding ki-netics of Leu and Ile substitutions at the same site, one isable to further probe the formation of detailed van derWaals contacts in a folding transition state, a luxury in anal-ysis not offered by conventional single Ala substitution ap-proach. More importantly, as opposed to Ala mutagenesis,which only reports on the formation of native structure sur-

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rounding a mutated side chain in the transition state (andonly by inference on the backbone conformation), the multi-ple substitution of surface-exposed positions can provide amore direct insight into the formation of a particular secon-dary structure motif in the folding transition state. In thiscase, if the backbone has adopted its native secondary struc-ture in the transition state, the DDG{?u values of the mu-tants made at a single site within the motif are expected tocorrelate with the changes in the local structure propensityassociated with the mutation. Also, by examining the corre-lation between DDG{?u and residue propensities to formnon-native secondary structure, it is possible to ascertainwhether a non-native backbone conformation is formed inthe transition state (Di Nardo et al. 2004). By characterizingthe folding kinetics of corresponding mutants in structurallyhomologous positions in different proteins, or different posi-tions within the same protein with varying tertiary contexts,the influence of context on folding kinetics can also beeasily investigated (Minor and Kim 1994a; Zarrine-Afsar etal. 2007).

Brønsted plots as a means to assess thefolding kinetics of multiple mutants made ata single site

A number of studies have used Brønsted plots (Fersht etal. 1994) to examine the kinetic properties of a large numberof proteins substituted at a single position (Mok et al. 2001;Northey et al. 2002b; Crespo et al. 2004; Anil et al. 2005;Simpson et al. 2006). The use of Brønsted plots for this pur-pose is based on the stipulation that if all of the mutationsused assess the same degree of structure formation in thetransition state there will be a simple relationship betweenthe folding rate constants and the changes in overall stabilityfor a set of mutants, and the kinetics data will follow theBrønsted equation (Fersht et al. 1994):

ln kf ¼ ln kof � bf �DDGf!u=RT

where k8f is the rate constant for folding of the wild typeprotein and bf is a constant related to the degree of structureformation in the transition state at the position of interest(see Northey et al. (2002b) for representative Brønstedplots). Any substitution violating the previously stated as-sumptions by (i) changing the structure of the folded and(or) unfolded state (assumptions 4 and 5), (ii) altering thefolding pathway of the protein (assumption 2), (iii) stabiliz-ing (or destabilizing) the folding transition state by introduc-ing new interactions (assumption 1), or (iv) modulating thestability through solvation effects (assumption 6) or non-native mechanisms (assumption 3) is expected to stand asan outlier in the Brønsted plot. Thus, linear Brønsted plotsfor multiple substitutions at a single position should indicatethat despite the non-conservative nature of the substitutionsused the assumptions necessary for rigorously interpretingthe F value of the position are valid.

The multiple substitution strategy as ameans to address the accuracy of F values

Determination of interpretable F values requires that theeffect of mutation on the overall stability DDGf?u (taken as

the reference in the denominator of F = DDG{?u /DDGf?u)be sufficiently large, and exceed the experimental error byseveral fold. Otherwise, the calculated F value may havean associated uncertainty on the order of, or even largerthan, the F value itself (for a description of errors associatedwith kinetics experiments see the Supplementary Materialaccompanying Zarrine-Afsar et al. (2008), and Zarrine-Afsarand Davidson (2004); Maxwell et al. 2005).

For each multiply substituted position, the slope ofDDG{?u as a function of DDGf?u is equivalent to an aver-age F value for the mutated position. For each mutant, a de-viation from the average F value can be calculatedaccording to |Fobs – Fave|. The Fobs for any substitution vio-lating the earlier assumptions is likely to exhibit a large de-viation from the average F value, and the substitutionpossessing the aberrant F value can be identified accord-ingly. By comparing the magnitude of F-value deviationwith the DDGf?u associated with the mutation, one can ob-tain an appropriate |DDGf?u| value required to obtain mean-ingful F values. This method was originally described by(Sanchez and Kiefhaber 2003c), where the authors proposeda minimum |DDGf?u| value of 1.7 kcal�mol–1 using a lim-ited data set of ~30 mutants made at only 3 positions in theFyn and Drk SH3 domains. In this work, the authors calcu-lated |Fobs – Fave| for each mutant as a function of DDGf?u,and subsequently determined an appropriate minimumDDGf?u value required to produce a deviation on the F

value that was acceptable within the experimental errorbounds. A potential caveat with this analysis is the use of alimited kinetic data set of multiply substituted positionsavailable at the time to gauge the accuracy of F values.

Interestingly, for most of the multiply substituted surface-exposed positions in the Fyn SH3 domain similar F valuesare obtained, as evidenced by strong correlation coefficientsobtained in Brønsted plots of E24, L29, R40, S41, T47, andN53 in the Fyn SH3 domain (Northey et al. 2002b; Zarrine-Afsar et al. 2007, 2008)(Arash Zarrine-Afsar, Sung Lun Lin,and Alan Davidson, unpublished observations), suggestingthat a minimum |DDGf?u| value of 1.7 kcal�mol–1 to obtainreliable F values is not always necessary. Nevertheless, foreach multiply mutated position, analysis of F-value devia-tion as described previously is a good practice, providing ameans to identify outliers exhibiting mutation-specific ef-fects that could complicate the interpretation of theF values.

The non-native character of foldingtransition states; the significance of themultiple amino acid substitution approach

In the standard interpretation of F values, as originally re-ported by the Fersht group (Matouschek et al. 1989) aminoacid side chains are assumed to stabilize the folding transi-tion state through the same interactions by which they stabi-lize the folded state. Thus, F values are expected to rangefrom 0 to unity, as discussed earlier. Inconsistent with thisbasic philosophy of F-value analysis, however, is a compre-hensive literature survey of the reported kinetics data (com-prising over 650 data points in 19 different proteins; SungLun Lin, Arash Zarrine-Afsar, and Alan Davidson, unpub-lished observations) that suggests that a large number

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(~25%) of the reported F values are non-canonical (F < 0or F > 1). Negative F values (~18%) may in some casesoriginate from non-native interactions that stabilize the fold-ing transition state (DDG{?u < 0), but destabilize the nativeinteractions in the folded state (DDGf?u > 0). In addition,mutagenesis of residues that make more favorable contactsin the folding transition state than in the folded state mayresult in F values that are greater than unity (F > 1), and asubset of these contacts have been shown to be non-nativein nature (Northey et al. 2002a).

In keeping with these considerations, non-native interac-tions have been identified experimentally in the foldingpathways of mutants of the SH3 domains of Src (Viguera etal. 2002) and Fyn (Di Nardo et al. 2004; Neudecker et al.2006; Zarrine-Afsar et al. 2008), as well as Hpr (Canet etal. 2003) and Im9 (Morton et al. 2007) proteins. These ob-servations collectively suggest that the structure of foldingtransition states could significantly deviate from the nativestate with many regions displaying non-native features. Con-sequently, the interactions that stabilize these states are notexclusively native-like in nature, and non-native interactionsin folding pathways are more widespread than currently rec-ognized. More interestingly, a subset of the studies that havereported non-native contacts in folding have also demon-strated that non-native interactions may assist in folding byaccelerating the folding rate (Di Nardo et al. 2004; Zarrine-Afsar et al. 2008), hereby confirming the predictions fromsimulations (Li et al. 2000; Treptow et al. 2002; Clementiand Plotkin 2004) and theory (Plotkin 2001) that non-nativeinteractions do not always stall folding, but can also speed itup under certain conditions. The existence of non-canonicalF values and the evidence that non-native interactions mayaid in folding by stabilizing the folding transition statesshould serve as a note of caution in the interpretation ofF values. More importantly, there is evidence that even can-onical F values (0 < F <1) may also result from the disrup-tion of non-native contacts (Cho et al. 2004), and this workvividly illustrates the danger associated with taking the re-sults of single FAla-value analysis at face value. In a similarvein, through the multiple amino acid substitutions of theG48 position in the Fyn SH3 domain a non-native backboneconformation was found to be a strong kinetic driving forcein the folding of this domain (Di Nardo et al. 2004), and the310 helix region in this protein was shown to be stabilizedby a non-native hydrophobic mechanism in the transitionstate (Zarrine-Afsar et al. 2008). In the latter case, a correla-tion was detected between the transition state stability andthe side chain hydrophobicity for all of the naturally occur-ring amino acids engineered at the 310 helix position. It isworth mentioning that single FAla-value analysis of the 310helix position would only suggest that the native structurein the 310 helix region was poorly formed in the transitionstate, providing no additional information regarding thenon-native hydrophobic stabilization of this helix, detectedonly via the multiple substitution approach (Zarrine-Afsar etal. 2008). This study highlights yet another advantage of themultiple substitution strategy in providing a wealth of infor-mation regarding folding pathways not readily obtainedthrough the single Ala substitution approach.

Probing the folding pathways in the absenceof denaturants: advances in NMR relaxationdispersion spectroscopy facilitate rigorousinterpretation of F values

If the folding equilibrium of a protein is maintained byexchange on the millisecond time-scale, and the denaturedand (or) intermediate (excited) states of that protein are suf-ficiently populated (approximately 0.5% or more, corre-sponding to a free energy difference of ~3.0 kcal mol–1),the chemical exchange to and from excited states results inline broadening of the NMR spectrum of the native (ground)state. Although the resonances of these excited states areusually undetectable, recent advances in NMR relaxationdispersion experiments allow an accurate reconstruction ofthe NMR spectra of these ‘‘invisible’’ states through quantify-ing the line broadening observed in the native state spectra(Korzhnev and Kay 2008; Neudecker et al. 2009). In addi-tion to the NMR chemical shifts for denatured (U) and (or)intermediate (I) states, a typical NMR relaxation dispersionexperiment also provides kinetic rate constants for the ex-change process. Accordingly, it has recently been demon-strated that NMR relaxation dispersion spectroscopy can beused to quantify F values (Neudecker et al. 2007) in the ab-sence of denaturants and without the use of stopped-flowbased methods. The availability of abundant site-specific,high-resolution, structural information in the form of NMRchemical shifts for the states in exchange allows identifica-tion of the excited state (e.g., as U or I), and significantlyfacilitates interpretation of F values because chemical shiftscan be used to monitor potential changes to the structure asa result of non-conservative mutagenesis. In addition to po-tential changes in the native state that could easily be probedby conventional NMR structure determination if a violationof assumption 5 is suspected, the chemical shifts extractedfrom NMR relaxation dispersion experiments also report onany residual structure in the denatured state, in violation ofassumption 4, discussed previously. While such informationis not directly available through conventional stopped-flowbased approaches, the multiple substitution strategy can pro-vide some insight into potential changes in the unfoldedstate structure as a result of mutagenesis. For example,changes in the kinetic m values for the folding rate (kf) ofcertain mutants (mkf; the slope of lnkf as a function of dena-turant concentration) can indicate changes in the unfoldedstate structure (Smith et al. 1994; Myers et al. 1995; Lopez-Hernandez et al. 1997) under certain conditions. However,such changes could also be due to other processes suchas Hammond (or anti-Hammond) behavior (Sanchez andKiefhaber 2003a, 2003b), in which mutations may alter theposition of the rate-limiting step with respect to the foldedstate on the reaction coordinate (Fersht et al. 1994).

Since violations of assumptions regarding transientlypopulated excited states such as folding transition states (as-sumptions 1–3) are more difficult to detect than those affect-ing the folded and unfolded states, one generally needs toresort to indirect evidence to assess the validity of these as-sumptions. In this regard, a detailed picture of the foldingpathway of the protein is highly desirable. Here again NMRrelaxation dispersion spectroscopy enjoys advantages overconventional stopped-flow methods, which only provide in-

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formation that is either averaged over all probes in the mol-ecule (e.g., circular dichroism) or restricted to where theprobes (e.g., tryptophan fluorescence) are located. First,NMR relaxation dispersion experiments can simultaneouslyprobe the folding kinetics at an abundance of site-specifi-cally resolved probes distributed all over the molecule (theNMR-active nuclei such as the backbone amide 15N and1HN, 13CO, 13Ca, 1Ha, methyl group 13C etc.). Second,although kinetic measurements as a function of denaturantconcentration are possible if desired (Zeeb and Balbach2005), the complete set of kinetic, thermodynamic, andstructural parameters of the exchange process is accessibleunder fully native conditions. As a consequence, NMR re-laxation dispersion experiments are very sensitive to low-populated intermediate states along the folding pathway,even to the so-called ‘‘unfolding intermediates’’ close to thenative structure, which become destabilized rapidly upon theaddition of denaturant, and are consequently undetectable instopped-flow based methods. Moreover, NMR detection andcharacterization of such intermediates can be facilitated bythe well-established measurement of hydrogen exchange ki-netics (Krishna et al. 2004). NMR relaxation dispersion pro-vides detailed kinetic, thermodynamic, and high-resolutionstructural information (in the form of chemical shifts and re-sidual dipolar couplings) about folding intermediates(Korzhnev and Kay 2008; Neudecker et al. 2009) and themethod is evolving rapidly. Obviously, detection and struc-tural characterization of intermediates adds considerable de-tail to our understanding of a particular folding pathway,including the sequence of formation of both native and non-native interactions (which can be identified on the basis ofthe chemical shifts data) along the reaction coordinate. Indi-rectly, this information may also aid in examining the valid-ity the assumptions regarding the rate-limiting transitionstates, 1–3, as discussed previously.

For example, NMR relaxation dispersion spectroscopy hasbeen used to probe the folding pathway of several mutantsof the Fyn and Abp1 SH3 domains (which share only 36%sequence identity) under native conditions (Korzhnev et al.2004, 2006). The extracted chemical shifts for the unfoldedstates of these proteins were found to be very similar tothose of random-coil peptides, suggesting that the denaturedstates of Fyn and Abp1 SH3 domains indeed have little re-sidual structure. Although conventional stopped-flow experi-ments to probe folding of the Fyn SH3 domain (Plaxco et al.1998; Northey et al. 2002a, 2002b; Di Nardo et al. 2004;Neudecker et al. 2006; Zarrine-Afsar et al. 2007) have beenconsistent with a two-state mechanism according to the ki-netic (i.e., single-exponential fluorescence recovery withoutany detectable rollover in the chevron plots)3 and calorimet-ric (i.e., the van’t Hoff enthalpy is equal to the calorimetricenthalpy) criteria (Fersht et al. 1994), the recent NMR relax-ation dispersion studies have revealed low-populated (1%–2%), on-pathway, folding intermediates (U$I$F) in all ofthe SH3 domain mutants investigated (Korzhnev et al.2004, 2006; Neudecker et al. 2006). In the case of the Fyn

SH3 A39V/N53P/V55L mutant, this apparent contradictionwas easily reconciled by noting that in the presence of evensmall amounts of denaturant the exchange between nativeand intermediate states becomes too rapid for detection, tothe point that the apparent two-state, stopped-flow datawould only report on the early transition state (U$I)(Neudecker et al. 2006). In addition to revealing the kinetic(exchange rates), thermodynamic (populations), and struc-tural (chemical shifts etc.) parameters, NMR relaxation dis-persion allowed F-value analysis of the three-state foldingpathway of the aforementioned Fyn SH3 domain mutant(Neudecker et al. 2007), with F values quantified for bothearly and late transition states and the intermediate state be-tween. Combined interpretation of all available NMR relax-ation dispersion data established that the central three-stranded b-sheet is already formed in the early transitionstate, whereas the rest of the domain was stabilized by non-native interactions in the on-pathway intermediate and didnot adopt its fully native conformation until after the latetransition state (Neudecker et al. 2007). These studies col-lectively highlight the power of NMR relaxation dispersionspectroscopy in providing an unprecedented level of detailin studies of millsecond time-scale protein folding.

Conclusions

Multiple substitution of protein folding transition states atsingle sites provides the best means to characterize thesetransient, yet important, states. In light of the fact that non-native interactions in folding pathways are more widespreadthan currently recognized, the results of single Ala mutagen-esis studies should be interpreted with caution. NMR relaxa-tion dispersion spectroscopy to quantify F values providesinformation regarding the structure of low-populated excitedstates in exchange; hereby significantly facilitating the inter-pretation of F values by offering a means to assess thevalidity of the assumptions required for a rigorous interpre-tation of F values. In principle, the two approaches can bereadily combined and NMR relaxation dispersion can beused to quantify F values of multiple substitutions at singlesites. This combined scheme, although laborious and time-consuming, provides a wealth of information regarding thefolding pathway that cannot be obtained through any othermethod presently used in the biophysical analysis of proteinfolding.

AcknowledgementsThe authors are indebted to Prof. Alan R. Davidson and

Prof. Lewis E. Kay (University of Toronto) for mentorshipand many years of support. The research on protein foldingin the Davidson and the Kay laboratories is supported bygrants from the Canadian Institutes of Health Research(CIHR) awarded to A.R.D and L.E.K. A.Z.-A. received sup-port from the Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) during his doctoral studies.S.L. is a trainee with the CIHR Training Grant in Protein

3 In the biophysical analysis of protein folding, the kinetic data (lnkobs versus [denaturant]) are often displayed in the form of a V-shaped‘‘chevron’’ plot. For two-state folders, the lnkobs for both folding and unfolding experiments has been shown to exhibit a linear dependenceon [denaturant], in the concentration range examined. Intermediates, populated at low [denaturant], slow down the observed folding rate,causing a curvature in the chevron plot referred to as a chevron roll over (Fersht 1999).

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Folding: Principles and Diseases, and P.N. acknowledges apost-doctoral fellowship from CIHR.

ReferencesAnil, B., Sato, S., Cho, J.H., and Raleigh, D.P. 2005. Fine structure

analysis of a protein folding transition state; distinguishing be-tween hydrophobic stabilization and specific packing. J. Mol.Biol. 354(3): 693–705. doi:10.1016/j.jmb.2005.08.054. PMID:16246369.

Canet, D., Lyon, C.E., Scheek, R.M., Robillard, G.T., Dobson,C.M., Hore, P.J., and van Nuland, N.A. 2003. Rapid formationof non-native contacts during the folding of HPr revealed byreal-time photo-CIDNP NMR and stopped-flow fluorescence ex-periments. J. Mol. Biol. 330(2): 397–407. doi:10.1016/S0022-2836(03)00507-2. PMID:12823977.

Cho, J.H., Sato, S., and Raleigh, D.P. 2004. Thermodynamics andkinetics of non-native interactions in protein folding: a singlepoint mutant significantly stabilizes the N-terminal domain ofL9 by modulating non-native interactions in the denatured state.J. Mol. Biol. 338(4): 827–837. doi:10.1016/j.jmb.2004.02.073.PMID:15099748.

Clementi, C., and Plotkin, S.S. 2004. The effects of nonnative in-teractions on protein folding rates: theory and simulation. Pro-tein Sci. 13(7): 1750–1766. doi:10.1110/ps.03580104. PMID:15215519.

Crespo, M.D., Platt, G.W., Bofill, R., and Searle, M.S. 2004. Con-text-dependent effects of proline residues on the stability andfolding pathway of ubiquitin. Eur. J. Biochem. 271(22): 4474–4484. doi:10.1111/j.1432-1033.2004.04392.x. PMID:15560788.

Dahiyat, B.I., and Mayo, S.L. 1997. Probing the role of packingspecificity in protein design. Proc. Natl. Acad. Sci. U.S.A.94(19): 10172–10177. doi:10.1073/pnas.94.19.10172. PMID:9294182.

Di Nardo, A.A., Korzhnev, D.M., Stogios, P.J., Zarrine-Afsar, A.,Kay, L.E., and Davidson, A.R. 2004. Dramatic acceleration ofprotein folding by stabilization of a nonnative backbone confor-mation. Proc. Natl. Acad. Sci. U.S.A. 101(21): 7954–7959.doi:10.1073/pnas.0400550101. PMID:15148398.

Fersht, A. 1999. Structure and mechanism in protein science: aguide to enzyme catalysis and protein folding. W.H. Freeman,New York, N.Y. pp. xxi, 631.

Fersht, A.R., and Sato, S. 2004. Phi-value analysis and the natureof protein-folding transition states. Proc. Natl. Acad. Sci.U.S.A. 101(21): 7976–7981. doi:10.1073/pnas.0402684101.PMID:15150406.

Fersht, A.R., Itzhaki, L.S., elMasry, N.F., Matthews, J.M., and Ot-zen, D.E. 1994. Single versus parallel pathways of protein fold-ing and fractional formation of structure in the transition state.Proc. Natl. Acad. Sci. U.S.A. 91(22): 10426–10429. doi:10.1073/pnas.91.22.10426. PMID:7937968.

Kim, C.A., and Berg, J.M. 1993. Thermodynamic beta-sheet pro-pensities measured using a zinc-finger host peptide. Nature,362(6417): 267–270. doi:10.1038/362267a0. PMID:8459852.

Korzhnev, D.M., and Kay, L.E. 2008. Probing invisible, low-popu-lated States of protein molecules by relaxation dispersion NMRspectroscopy: an application to protein folding. Acc. Chem. Res.41(3): 442–451. doi:10.1021/ar700189y. PMID:18275162.

Korzhnev, D.M., Salvatella, X., Vendruscolo, M., Di Nardo, A.A.,Davidson, A.R., Dobson, C.M., and Kay, L.E. 2004. Low-popu-lated folding intermediates of Fyn SH3 characterized by relaxa-tion dispersion NMR. Nature, 430(6999): 586–590. doi:10.1038/nature02655. PMID:15282609.

Korzhnev, D.M., Neudecker, P., Zarrine-Afsar, A., Davidson, A.R.,

and Kay, L.E. 2006. Abp1p and Fyn SH3 domains fold throughsimilar low-populated intermediate states. Biochemistry, 45(34):10175–10183. doi:10.1021/bi0611560. PMID:16922492.

Krishna, M.M., Hoang, L., Lin, Y., and Englander, S.W. 2004. Hy-drogen exchange methods to study protein folding. Methods,34(1): 51–64. doi:10.1016/j.ymeth.2004.03.005. PMID:15283915.

Li, L., Mirny, L.A., and Shakhnovich, E.I. 2000. Kinetics, thermo-dynamics and evolution of non-native interactions in a proteinfolding nucleus. Nat. Struct. Biol. 7(4): 336–342. doi:10.1038/74111. PMID:10742180.

Lopez-Hernandez, E., Cronet, P., Serrano, L., and Munoz, V. 1997.Folding kinetics of Che Y mutants with enhanced native alpha-helix propensities. J. Mol. Biol. 266(3): 610–620. doi:10.1006/jmbi.1996.0793. PMID:9067614.

Martınez, J.C., and Serrano, L. 1999. The folding transition statebetween SH3 domains is conformationally restricted and evolu-tionarily conserved. Nat. Struct. Biol. 6(11): 1010–1016. doi:10.1038/14896. PMID:10542091.

Matouschek, A., Kellis, J.T., Jr., Serrano, L., and Fersht, A.R.1989. Mapping the transition state and pathway of protein fold-ing by protein engineering. Nature, 340(6229): 122–126. doi:10.1038/340122a0. PMID:2739734.

Maxwell, K.L., Wildes, D., Zarrine-Afsar, A., De Los Rios, M.A.,Brown, A.G., Friel, C.T., et al. 2005. Protein folding: defining a‘‘standard’’ set of experimental conditions and a preliminary ki-netic data set of two-state proteins. Protein Sci. 14(3): 602–616.doi:10.1110/ps.041205405. PMID:15689503.

Minor, D.L., Jr., and Kim, P.S. 1994a. Context is a major determi-nant of beta-sheet propensity. Nature, 371(6494): 264–267.doi:10.1038/371264a0. PMID:8078589.

Minor, D.L., Jr., and Kim, P.S. 1994b. Measurement of the beta-sheet-forming propensities of amino acids. Nature, 367(6464):660–663. doi:10.1038/367660a0. PMID:8107853.

Mok, Y.K., Elisseeva, E.L., Davidson, A.R., and Forman-Kay, J.D.2001. Dramatic stabilization of an SH3 domain by a single sub-stitution: roles of the folded and unfolded states. J. Mol. Biol.307(3): 913–928. doi:10.1006/jmbi.2001.4521. PMID:11273710.

Morton, V.L., Friel, C.T., Allen, L.R., Paci, E., and Radford, S.E.2007. The effect of increasing the stability of non-native interac-tions on the folding landscape of the bacterial immunity proteinIm9. J. Mol. Biol. 371(2): 554–568. doi:10.1016/j.jmb.2007.05.010. PMID:17574573.

Myers, J.K., Pace, C.N., and Scholtz, J.M. 1995. Denaturant m va-lues and heat capacity changes: relation to changes in accessiblesurface areas of protein unfolding. Protein Sci. 4(10): 2138–2148. doi:10.1002/pro.5560041020. PMID:8535251.

Neudecker, P., Zarrine-Afsar, A., Choy, W.Y., Muhandiram, D.R.,Davidson, A.R., and Kay, L.E. 2006. Identification of a col-lapsed intermediate with non-native long-range interactions onthe folding pathway of a pair of Fyn SH3 domain mutants byNMR relaxation dispersion spectroscopy. J. Mol. Biol. 363(5):958–976. doi:10.1016/j.jmb.2006.08.047. PMID:16989862.

Neudecker, P., Zarrine-Afsar, A., Davidson, A.R., and Kay, L.E.2007. Phi-value analysis of a three-state protein folding pathwayby NMR relaxation dispersion spectroscopy. Proc. Natl. Acad.Sci. U.S.A. 104(40): 15717–15722. doi:10.1073/pnas.0705097104. PMID:17898173.

Neudecker, P., Lundstrom, P., and Kay, L.E. 2009. Relaxation dis-persion NMR spectroscopy as a tool for detailed studies of pro-tein folding. Biophys. J. 96(6): 2045–2054. doi:10.1016/j.bpj.2008.12.3907. PMID:19289032.

Northey, J.G., Di Nardo, A.A., and Davidson, A.R. 2002a. Hydro-phobic core packing in the SH3 domain folding transition state.

Zarrine-Afsar et al. 237

Published by NRC Research Press

Bio

chem

. Cel

l Bio

l. D

ownl

oade

d fr

om w

ww

.nrc

rese

arch

pres

s.co

m b

y G

eorg

e M

ason

Uni

vers

ity o

n 05

/05/

13Fo

r pe

rson

al u

se o

nly.

Nat. Struct. Biol. 9(2): 126–130. doi:10.1038/nsb748. PMID:11786916.

Northey, J.G., Maxwell, K.L., and Davidson, A.R. 2002b. Proteinfolding kinetics beyond the phi value: using multiple aminoacid substitutions to investigate the structure of the SH3 domainfolding transition state. J. Mol. Biol. 320(2): 389–402. doi:10.1016/S0022-2836(02)00445-X. PMID:12079394.

Permyakov, S.E., Makhatadze, G.I., Owenius, R., Uversky, V.N.,Brooks, C.L., Permyakov, E.A., and Berliner, L.J. 2005. How toimprove nature: study of the electrostatic properties of the sur-face of alpha-lactalbumin. Protein Eng. Des. Sel. 18(9): 425–433. doi:10.1093/protein/gzi051. PMID:16093284.

Plaxco, K.W., Guijarro, J.I., Morton, C.J., Pitkeathly, M., Camp-bell, I.D., and Dobson, C.M. 1998. The folding kinetics andthermodynamics of the Fyn-SH3 domain. Biochemistry, 37(8):2529–2537. doi:10.1021/bi972075u. PMID:9485402.

Plotkin, S.S. 2001. Speeding protein folding beyond the G(o)model: how a little frustration sometimes helps. Proteins, 45(4):337–345. doi:10.1002/prot.1154. PMID:11746681.

Sanchez, I.E., and Kiefhaber, T. 2003a. Hammond behavior versusground state effects in protein folding: evidence for narrow freeenergy barriers and residual structure in unfolded states. J. Mol.Biol. 327(4): 867–884. doi:10.1016/S0022-2836(03)00171-2.PMID:12654269.

Sanchez, I.E., and Kiefhaber, T. 2003b. Non-linear rate-equilibriumfree energy relationships and Hammond behavior in proteinfolding. Biophys. Chem. 100(1-3): 397–407. doi:10.1016/S0301-4622(02)00294-6. PMID:12646379.

Sanchez, I.E., and Kiefhaber, T. 2003c. Origin of unusual phi-va-lues in protein folding: evidence against specific nucleationsites. J. Mol. Biol. 334(5): 1077–1085. doi:10.1016/j.jmb.2003.10.016. PMID:14643667.

Sanchez-Ruiz, J.M., and Makhatadze, G.I. 2001. To charge or notto charge? Trends Biotechnol. 19(4): 132–135. doi:10.1016/S0167-7799(00)01548-1. PMID:11250029.

Schweiker, K.L., Zarrine-Afsar, A., Davidson, A.R., and Makha-tadze, G.I. 2007. Computational design of the Fyn SH3 domainwith increased stability through optimization of surface chargecharge interactions. Protein Sci. 16(12): 2694–2702. doi:10.1110/ps.073091607. PMID:18029422.

Simpson, E.R., Meldrum, J.K., and Searle, M.S. 2006. Engineeringdiverse changes in beta-turn propensities in the N-terminal beta-hairpin of ubiquitin reveals significant effects on stability and

kinetics but a robust folding transition state. Biochemistry,45(13): 4220–4230. doi:10.1021/bi052495g. PMID:16566596.

Smith, C.K., Withka, J.M., and Regan, L. 1994. A thermodynamicscale for the beta-sheet forming tendencies of the amino acids.Biochemistry, 33(18): 5510–5517. doi:10.1021/bi00184a020.PMID:8180173.

Treptow, W.L., Barbosa, M.A.A., Garcia, L.G., and Pereira de Ara-ujo, A.F. 2002. Non-native interactions, effective contact order,and protein folding: a mutational investigation with the energeti-cally frustrated hydrophobic model. Proteins, 49(2): 167–180.doi:10.1002/prot.10166. PMID:12210998.

Viguera, A.R., Vega, C., and Serrano, L. 2002. Unspecific hydro-phobic stabilization of folding transition states. Proc. Natl.Acad. Sci. U.S.A. 99(8): 5349–5354. doi:10.1073/pnas.072387799. PMID:11959988.

Xu, J., Baase, W.A., Baldwin, E., and Matthews, B.W. 1998. Theresponse of T4 lysozyme to large-to-small substitutions withinthe core and its relation to the hydrophobic effect. Protein Sci.7(1): 158–177. PMID:9514271.

Zarrine-Afsar, A., and Davidson, A.R. 2004. The analysis of pro-tein folding kinetic data produced in protein engineering experi-ments. Methods, 34(1): 41–50. doi:10.1016/j.ymeth.2004.03.013.PMID:15283914.

Zarrine-Afsar, A., Larson, S.M., and Davidson, A.R. 2005. The fa-mily feud: do proteins with similar structures fold via the samepathway? Curr. Opin. Struct. Biol. 15(1): 42–49. doi:10.1016/j.sbi.2005.01.011. PMID:15718132.

Zarrine-Afsar, A., Dahesh, S., and Davidson, A.R. 2007. Proteinfolding kinetics provides a context-independent assessment ofbeta-strand propensity in the Fyn SH3 domain. J. Mol. Biol.373(3): 764–774. doi:10.1016/j.jmb.2007.07.059. PMID:17850820.

Zarrine-Afsar, A., Wallin, S., Neculai, A.M., Neudecker, P., Ho-well, P.L., Davidson, A.R., and Chan, H.S. 2008. Theoreticaland experimental demonstration of the importance of specificnonnative interactions in protein folding. Proc. Natl. Acad. Sci.U.S.A. 105(29): 9999–10004. doi:10.1073/pnas.0801874105.PMID:18626019.

Zeeb, M., and Balbach, J. 2005. NMR spectroscopic characteriza-tion of millisecond protein folding by transverse relaxation dis-persion measurements. J. Am. Chem. Soc. 127(38): 13207–13212. doi:10.1021/ja051141+. PMID:16173748.

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