6448 | Phys. Chem. Chem. Phys., 2014, 16, 6448--6459 This journal is© the Owner Societies 2014

Cite this:Phys.Chem.Chem.Phys.,

2014, 16, 6448

Diffuse transition state structure for the unfoldingof a leucine-rich repeat protein

Sadie E. Kelly,a Georg Meisl,b Pamela J. E. Rowling,†b Stephen H. McLaughlin,c

Tuomas Knowles*b and Laura S. Itzhaki†*b

Tandem-repeat proteins, such as leucine-rich repeats, comprise arrays of small structural motifs that

pack in a linear fashion to produce elongated architectures. They lack contacts between residues that

are distant in primary sequence, a feature that distinguishes them from the complex topologies of

globular proteins. Here we have investigated the unfolding pathway of the leucine-rich repeat domain of

the mRNA export protein TAP (TAPLRR) using F-value analysis. Whereas most of the tandem-repeat

proteins studied to date have been found to unfold via a polarised mechanism in which only a small,

localised number of repeats are structured in the transition state, the unfolding mechanism of TAPLRR is

more diffuse in nature. In the transition state for unfolding of TAPLRR, three of the four LRRs are highly

structured and non-native interactions are formed within the N-terminal a-helical cap and the first LRR.

Thus, the a-helical cap plays an important role in which non-native interactions are required to provide

a scaffold for the LRRs to pack against in the folding reaction.

Introduction

Tandem repeat proteins, such as ankyrin, tetratricopeptide(TPR), HEAT and leucine-rich repeats, are composed of tandemarrays of 20–40 amino acid structural motifs that pack togetherin a roughly linear fashion to produce elongated non-globulararchitectures. They present extended surfaces for molecularrecognition and thereby mediate diverse functions within thecell. Unlike globular proteins, which have complex topologiesinvolving many interactions between sequence-distant residues,tandem repeat proteins are stabilised solely by short-rangeinteractions between residues close in sequence. Therefore theformation of the structures of these elongated proteins duringthe folding process can be studied in a segmented fashion.1–11

Leucine-rich repeat (LRR) proteins are characterised byrepetition of a B20 amino acid sequence motif with theconsensus sequence Lin–Xout–Lin–Xout–Lin–Xout–Xout–Nin whereL is a leucine residue, X is any amino acid and N is anasparagine residue. The subscripts ‘‘in’’ and ‘‘out’’ refer towhether the side chain of the residue points in to the hydro-phobic core of the structure or out to the solvent. The repeats

form a concave surface of short (6 or 7 residue) b-strands anda concave surface of a-helices that run approximately anti-parallel to the b-strands (Fig. 1). The consensus sequencecorresponds to the b-strand of each repeat, with the leucineresidues making stacking interactions that maintain the hydro-phobic core of the domain. Whereas ankyrin repeats and TPRshave been extensively characterised,1–3,5–18 much less is knownabout the folding mechanisms of LRR proteins.19–23 Residue-specific information about folding mechanisms, obtained usingthe F-value approach, is available for only one LRR protein todate, Internalin B.21 The results showed that the N-terminala-helical capping motif polarises the folding mechanism byacting as a nucleus upon which the repeats assemble.

The LRR domain of the nuclear export factor TAP (referredto subsequently as TAPLRR) is responsible for binding theconstitutive transport element of the RNA.24,25 It comprisesfour tandem LRRs and an a-helical cap at the N-terminus. Herewe have investigated the unfolding mechanism of TAPLRRusing F-value analysis, in which single-site, conservative sub-stitutions are made throughout the structure and the effects onthe folding/unfolding kinetics are compared with those on thestability, thereby allowing structural information about thetransition-state ensemble to be obtained. For all natural repeatproteins studied to date, structure in the folding/unfoldingtransition states has been found to be localised to a smallsubset of the repeats.1,6,11,26 In contrast, the transition statestructure for TAPLRR is rather diffusely distributed, with inter-actions being well formed in three of the four repeats. Asobserved for Internalin B,21 the a-helical cap of TAPLRR

a MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road,

Cambridge CB2 0XZ, UKb Department of Chemistry, University of Cambridge, Lensfield Road,

Cambridge CB2 1EW, UK. E-mail: lsi10@cam.ac.uk, tpjk2@cam.ac.ukc MRC Laboratory of Molecular Biology, Francis Crick Avenue,

Cambridge Biomedical Campus, Cambridge CB2 0QH, UK

† Current address: Department of Pharmacology, University of Cambridge,Tennis Court Road, Cambridge CB2 1PD, UK.

Received 14th November 2013,Accepted 10th February 2014

DOI: 10.1039/c3cp54818j

www.rsc.org/pccp

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appears to play an important role in the folding. However, in thecase of TAPLRR, non-classical F-values indicate that residues inthe cap form non-native interactions in the transition state.

ResultsTAPLRR unfolds at equilibrium via an apparent two-statemechanism

The TAPLRR construct used in this work consists of179 residues. A non-cleavable hexa-Histidine tag at theC-terminus is followed by the N-terminal helical cap comprising49 residues, the LRR domain is 104 residues, and the unstruc-tured C-terminal tail is 20 residues. The stability of TAPLRR was

measured using the fluorescence of the single tryptophanresidue (W321 located in b4 of LRR3) and by circular dichroism(CD). The fluorescence-monitored urea denaturation curve canbe fitted to a two-state equation, giving an m-value (a constantof proportionality that is related to the change in solventexposure of hydrophobic side chains upon unfolding) of3.21 � 0.25 kcal mol�1 M�1 and a midpoint of unfolding (D50%)of 1.64 � 0.02 M urea. The same results were obtained when thedata were plotted at different emission wavelengths. The free

energy of unfolding in water DGH2OD�N

� �was 5.26� 0.42 kcal mol�1.

The refolding denaturation curve superimposes on the unfoldingdenaturation curve, with the same midpoint and m-valuewithin error, indicating that the unfolding of TAPLRR isreversible in these experimental conditions. The denaturationcurve monitored by ellipticity at 222 nm to probe a-helicalstructure can also be fitted to a two-state transition but them-value is different from that obtained by fluorescence. If thefluorescence- and CD-monitored denaturation curves are fitted byfixing m to the weighted-average value of the wild type and mutantsas monitored by fluorescence (2.66 � 0.14 kcal mol�1 M�1), themidpoints of unfolding obtained are the same within error(1.60 � 0.03 M and 1.64 � 0.02 M for the fluorescence andCD data, respectively) (Fig. 2A). Therefore the equilibriumunfolding of TAPLRR can be approximated to a two-statemodel. The m-value is similar to that of another small LRRdomain, Internalin B.20

Both the refolding kinetics and the unfolding kinetics ofTAPLRR are biphasic

The refolding and unfolding kinetics of wild-type TAPLRR weremeasured by stopped-flow fluorescence. The kinetic traces forboth reactions were not well described by single exponentialphases (see Fig. 2D) but they could be fitted to the sum of twoexponential phases (Fig. 2C, E and F). The fast phase ofunfolding accounts for B85% of the total amplitude and theslow phase for B15%. The fast phase of refolding accounts forB80% of the total amplitude and the slow phase for B20%.The urea dependence of the observed rate constants and ampli-tudes is shown in Fig. 3B. Formation of oligomers or aggregatescan retard the folding reaction; however for TAPLRR no proteinconcentration dependence of the rate constants of refolding ortheir relative amplitudes was observed in the range of 0.5 mM to30 mM, indicating that oligomerisation is not the cause of the slowrefolding phase. Alternatively, the slow refolding phase could belimited by proline isomerisation (TAPLRR has nine prolineresidues), although the presence of the peptidyl–prolyl cis–transisomerase cyclophilin did not speed up the refolding kinetics,or it could correspond to one step of a multi-step reaction.

The urea dependence of the rate constant of unfoldingclearly shows downward curvature over the wide range of ureaconcentrations assayed. Similar non-linearity has been observed formany other proteins and it can be rationalised as follows: unfoldingcan be viewed as taking place over a broad energy barrier with asmooth movement across it as the denaturant concentrationis increased, in accordance with Hammond behaviour.27,28

Fig. 1 Structure of the LRR domain of TAP (TAPLRR). (A) The four leucine-rich repeats are shown coloured from N- to C-terminus in blue (repeat 1),turquoise (repeat 2), gold (repeat 3) and red (repeat 4). The N-terminalhelical cap is shown in grey. The side chain of the single tryptophan residue(W321) is shown in green. The side chains of the residues mutated in thisstudy are shown in black. (B) Structure of repeat 3 of TAPLRR showing theside chains of all the residues, coloured according to their properties.Leucine residues are coloured black. Polar, hydrophilic residues (Asn, Ser)are in grey, non-polar hydrophobic residues (Ile, Trp, Gly) are in gold,acidic residues (Asp, Glu) are coloured purple, basic residues (Lys, Arg)are in green.

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Fig. 2 Equilibrium and kinetic folding of wild-type TAPLRR. (A) Denaturation curves for wild-type TAPLRR, monitored by fluorescence (black) and CD(blue). The data were fitted to a two-state equation, represented by the solid line in the same colour. (B) and (C) Kinetic traces for refolding at 0.5 M and1.6 M urea, the solid line is the fit of a double exponential. (D) Kinetic trace for refolding at 1.6 M urea, the solid line is the fit of a single exponential, withlinear instrumental drift. Clearly this does not reproduce the observed kinetic trace and hence justifies the fitting of a double exponential. (E) and(F) Kinetic trace for unfolding at 5 M and 8.6 M urea. In both cases the solid black line is a fit to a double exponential decay. In figures (B) to (F) theresiduals are shown below the plot, normalised by the maximum fluorescence value of the corresponding kinetic trace; the fluorescence intensityis in arbitrary units.

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The data can be fitted to a quadratic equation to describe thisbehaviour:

ln ku½U� ¼ ln kH2Ou þmu½U� þm�½U�2 (1)

where kH2Ou is the rate constant of unfolding in water, mu is a

constant that is proportional to the increase in solvent accessible

surface area between the native state and the transition state,and m* is the denaturant dependence of mu. The fitting of thekinetic data is discussed in detail in the next section.

Modelling of the kinetic data

A double exponential decay, as observed for both the refoldingand unfolding kinetic traces, is indicative of a system involving

Fig. 3 Fitting to microscopic rates of folding in 3-state mechanisms. (A) Best fit to the off-pathway (top) and on-pathway (bottom) model. Whereas theon-path fit is comparable to the triangular fit in B, the off-path fit is significantly worse. (B) Best global fit of the rates (top) and amplitudes (bottom) to thetriangular mechanism. The rates are shown in grey for the major phase and blue for the minor phase; the solid line in the same colour is the best fit.(C) Fits of the curvature in the urea dependence of the logarithms of the rate constants, due to Hammond behaviour, for both the unfolding arm and therefolding arm of the major phase. (D) A schematic of the energy landscape of folding. This schematic is for illustration purposes only, and the heights ofthe barriers and energies of the species are not intended to accurately reflect the experimentally observed rates. (E) The microscopic rates and their ureadependences for the best fits as shown above. Note that the rates to the intermediate for the triangular case are very small compared to the rates fromthe intermediate, suggesting a high-energy intermediate, which is in accordance with its absence in the equilibrium denaturation curves. Due to the smallrates these transitions have little effect on the overall behaviour so their rates and m values may only be accurate to within an order of magnitude.

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three species.29 The most general model for a three-speciessystem is a triangular mechanism, wherein each of the speciesis connected to the other two by a forward and reverse rate.In the limit that the rates between two species are vanishinglysmall, the more common cases of on-pathway intermediate (I)(if the rate between the native state (N) and the denaturedstate (D) is small) and off-pathway intermediate (if the ratebetween native state and intermediate is small) are obtained.In a F-value analysis the changes in the folding and unfoldingrates upon mutation of specific residues are used to inferthe structure in the transition state. For systems involvingthree interconnected species, there will in general be threetransition states, which define six rate constants connectingthe three species, and for a meaningful F-value analysisone needs to ensure that only one transition state contributesto the results and establish which species it connects. One cansolve the kinetic equations for a general system of threeinterconverting species and relate the results to observables29

such as fluorescence. The observed rates are, in general,determined by a combination of all the microscopic rateconstants, but often the larger of the two observed rates isfound, to a good approximation, to be dominated by onemicroscopic rate. In that case the observed rate constant canbe assigned to a specific microscopic rate and thereby a specifictransition state in the system, which is crucial if a F-valueanalysis is to be attempted. The rate constant for the minorphase often shows a dependence on several microscopic ratesand its interpretation in terms of specific reaction steps is lessstraightforward.

In an effort to delineate the folding pathway, a doubleexponential was used to fit the time courses of the fluorescencefor the folding and unfolding experiments, giving two ampli-tudes and two observed rate constants at each denaturantconcentration; these observed data were fitted to three differentmodels: on-pathway intermediate, off-pathway intermediateand a triangular mechanism, as shown in Fig. 3A and B. As iscommonly the case, we assumed that the natural log of themicroscopic rates (ln(ki)) varies linearly with denaturantconcentration, with slope mi (refer to the Methods sectionfor the detailed equations). Using the equations derived inref. 29 and as applied in ref. 30 and 31, this assumptionthen allows us to fit the full dataset with 8 free parameters(4 rates, 4 slopes) in the case of the on- and off-pathwaymechanisms, or 10 free parameters in the case of the triangularmechanism.

The best fits of the data to the linear mechanisms are shownin Fig. 3A. An off-pathway mechanism can be discarded whenconsidering the wild-type TAPLRR data, on the grounds that itgives a poor fit to the exponents. The fits to the on-pathwaymechanism were as good as the fits to the more generaltriangular mechanism, but the on-pathway mechanism couldbe discarded by looking at the behaviour of the minor phaseupon mutation (discussed further in the next sections; see alsoFig. 6): in the on-pathway fits the major rate was, to a goodapproximation, determined only by the rate constants betweenthe intermediate and the native state, whereas the minor phase

was determined by the rate constants between the denaturedstate and the intermediate. This arrangement of the rate-determining transition states in series is not consistent withthe fact that the major and minor phases respond in a verysimilar way to mutation. Consider, for example, the mutantI264V (Fig. 6): the fact that the observed rates for the minorunfolding phase of this mutant are much faster than those ofwild type suggests that the structure is fully broken in thetransition state going from I and D, whereas the faster rates forthe major unfolding phase of this mutant compared with wildtype suggest that the structure is fully broken in the transitionstate going from N to I. It is not possible in a linear on-pathway-intermediate mechanism for structure to be fully broken twice.Therefore a triangular mechanism must instead be considered.A global fit of the observed rates and amplitudes to thetriangular mechanism is shown in Fig. 3B.

The parameters obtained in the fits are summarised in thetable in Fig. 3E, and in the triangular mechanism the majorphase is determined by the rates k3 (at low denaturant) andk4 (at high denaturant) over the entire range of denaturantconcentrations. This means that the dominant transition stateremains the same at different denaturant concentrations,namely the transition state between D and N, making aF-value analysis possible using the major phase. The minorphase is determined by rates k1 and k5 and hence the behaviourupon mutation can be explained if the intermediate and thetransition states leading to it (TSNI and TSID) are similar instructure to the transition state (TSND) determining the majorphase (see Fig. 3D).

As was mentioned above, the urea dependence of thelogarithm of the rate of the major phase displays negativecurvature. This can not be explained assuming a linear depen-dence of ln(ki) on the denaturant concentration and the deviationfrom the experimental data is evident in the fits. We thereforeassumed a quadratic dependence of the two rates dominating themajor phase, as given in eqn (1), and fitted the major phase asshown in Fig. 3C. Only the major phase was fitted with thisquadratic dependence, as including this factor in the fitting ofboth phases would require an additional parameter for each rateand therefore would result in an unfeasibly large number offitting parameters. The fits to the whole data set in Fig. 3B shouldtherefore be considered mainly as a means of relating themacroscopically observed phases to specific microscopic stepson the folding pathway. A quadratic fit to the major phase wasused for all mutants in order to extract the rates and thereby theF-values of the major phase. The F-value analysis of TAPLRR isdescribed below.

Single-site substitutions greatly destabilise TAPLRR

A first round of mutations was designed at solvent-exposedsites throughout the structure. Given the low stability of theTAPLRR, it was hoped that these mutations would perturb thestructure sufficiently to allow F-value analysis but not so muchas to cause unfolding or misfolding. The parameters obtainedfrom fitting the equilibrium unfolding data of the mutantsto a two-state model are shown in Table 1. Four proteins

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(M216L, V287A, S339A and L349I) were expressed in this firstround of mutagenesis. However, none of them had significanteffects on the equilibrium and kinetics of the protein, andhence they could not be used for F-value analysis.

Next, mutations were made at sites in the hydrophobic core.Many of these sites are leucine residues, and mutation of asubset of these (L296, L298, L303, L309, all located in repeat 3)to alanine was found to destabilise the protein to such anextent that the mutants could not be obtained in a solublefolded form in sufficient quantities. A similar sensitivity tomutation was observed previously for the LRR domain ofInternalin B.21 Mutation to alanine of L267 in repeat 2, L293in repeat 3 and L348 in repeat 4, was attempted, as well asmutation to glycine of A290 in repeat 2, but only very smallquantities of the proteins could be produced in soluble form.The following were successfully expressed and purified andfound to be folded like the wild type, as judged by gel filtrationand CD: two mutations were made at equivalent positions inrepeat 2 (I264V) and repeat 3 (L315A) (Fig. 4), and five muta-tions were made at equivalent positions in the loop at the endof each repeat between the a-helix and the b-sheet, in repeat 1(L267I), repeat 2 (L293I and L293V), repeat 3 (L317I) and repeat4 (L348I) (Fig. 4). In common with other LRR proteins TAPLRRhas a helical cap at one end that is thought to protect theoverall repeat structure by shielding the hydrophobic core. Thecap in TAPLRR is at the N-terminus and comprises a longa-helix followed by a b-sheet and a shorter a-helix; two mutationswere made in the N-terminal cap (L212V and L238I (Fig. 4)). Allbut two of the hydrophobic core mutations showed changes instability that are within the range compatible with accurate

F-value determination i:e: DDGH2OD�N 4 0:5 kcal mol�1

� �. For

the two exceptions, L238I and L267I, the values of DDGH2OD�N

were very small but the mutations had significant effects on

the kinetics. These two mutants are therefore also included inthe discussion below.

U-value analysis

The V-shaped plots for the major phase in the kinetics of theTAPLRR mutants are shown in Fig. 5. Curvature is evident inthe unfolding and refolding limbs for most of the mutants,as was observed for the wild type. To calculate the F values,we fitted the unfolding limbs, outside the transition region, to aquadratic equation (eqn (1)). The values of mu and m* obtainedfor the mutants were similar to those of the wild type, indicat-ing that the mutations do not cause a gross change in thetransition state structure. To minimise the errors, the data werethen refitted to a quadratic equation using a weighted-averagevalue of the curvature, calculated from wild type and all themutants. F values were determined at a urea concentrationof 5.5 M, which is in the middle of the urea range used for theunfolding studies, in order to avoid the need for a longextrapolation back to 0 M denaturant and thereby minimisethe errors. The kinetic parameters, including the F values, arelisted in Table 2.

L293V in repeat 2 and L348I in repeat 4 have F values closeto 1, indicating that these regions are fully structured in thetransition state, and residues in repeat 3 have high fractionalF values, indicating that this region is also highly structured inthe transition state. The N-terminal region of the protein is lesswell structured in the transition state: L212V in the N-terminalcap has a fractional F value and I264V in repeat 1 has a F valueof 0, indicating that this region is unstructured in the transitionstate. Interestingly, the mutation L267I (repeat 1) has a negli-gible effect on the equilibrium stability but its unfoldingkinetics is significantly slower than that of the wild-type pro-tein. The consequence of this behaviour is a non-classical

Table 1 Equilibrium denaturation parameters for wild type and mutants of TAPLRR

Protein Location Location of interacting residues m-value (kcal mol�1 M�1) [D]50% (M) DDGH2OU�N kcal mol�1

� �WT 3.21 � 0.25 1.64 � 0.02 —L212V a1 cap LRR1 — 1.08 � 0.11a 1.38 � 0.31M216L a1 cap LRR1 2.56 � 0.14 1.70 � 0.02 �0.27 � 0.10L238I a2 cap Cap 3.44 � 0.44 1.51 � 0.03 0.35 � 0.13I264V a3-b2 strand LRR1 Cap 3.40 � 0.21 1.41 � 0.02 0.64 � 0.10L267I a3-b2 strand LRR1 Cap LRR2 2.27 � 0.23 1.52 � 0.05 0.11 � 0.11V287A a4 LRR2 LRR3 2.66 � 0.24 1.54 � 0.03 0.16 � 0.11L293I a4-b3 strand LRR2 LRR1 LRR3 2.92 � 0.23 1.33 � 0.03 0.80 � 0.12L293V a4-b3 strand LRR2 LRR1 LRR3 — 1.07 � 0.06a 1.41 � 0.19L315A a5-b4 strand LRR3 LRR2 LRR4 — 0.98 � 0.09a 1.65 � 0.27L317I a5-b4 strand LRR3 LRR2 LRR4 2.52 � 0.22 1.26 � 0.04 0.85 � 0.12S339A a6 LRR4 LRR4 2.85 � 0.55 1.73 � 0.06 �0.32 � 0.18L348I a6-b5 strand LRR4 LRR3 C-terminus 2.88 � 0.41 1.42 � 0.05 0.53 � 0.14L349I b5 LRR4 LRR3 2.38 � 0.30 1.60 � 0.05 �0.05 � 0.13

The m-values and midpoints of unfolding ([U]50%) listed are those obtained by fitting the individual denaturation curves to a two-state equation. Theweighted-average of the m-values of wild type and those mutants with long pre-transition baselines in the denaturation curves (hmi = 2.66 � 0.14)was then used to refit the denaturation curves. The midpoints of unfolding obtained from these fits were used to calculate the changes in the

free energy of unfolding upon mutation, DDGH2OD�N ¼ hmi ½U�

50%wild type � ½U�

50%mutant

� �. The errors are the standard deviations from the fits of the

data. a The pre-transition baseline was absent or very short for three highly destabilising mutations, L212V, L293V and L315A, resulting in poorfits of the data. Therefore, for these mutants the values of [U]50% listed are those obtained by fitting the denaturation curves with the m-valuefixed at hmi.

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F value of greater than 1. Likewise, the mutation L238I (in thecap) has a negligible effect on the equilibrium stability butsignificantly speeds up the unfolding kinetics. The consequenceis a non-classical F value of less than 0. These non-classical

F values indicate the presence of non-native interactions in thetransition state.

DiscussionThe thermodynamic stability of TAPLRR is very sensitive tomutation of the consensus leucine residues

The low thermodynamic stability of TAPLRR DDGH2OD�N ¼

�

5:3 kcal mol�1Þ limited the number of mutant proteins thatcould be expressed in soluble form. In the hydrophobic core,the conserved leucine residues of repeat 3 were first selected formutation to alanine but only one mutant could be expressed ina soluble form to sufficient quantities (L315A). The largetruncation of the leucine side chain to alanine may disruptthe core packing interactions to such an extent as to causeunfolding of the protein. A similar sensitivity to mutation wasobserved for the small LRR domain of Internalin B.21 Moreconservative mutations were therefore made in the otherrepeats. Conversely, many of the substitutions at surface sitesin the LRR structure could be expressed in a soluble form butdid not destabilise the structure sufficiently to allow F-valueanalysis.

U-value analysis of the major kinetic unfolding phase shows amore diffusely structured unfolding transition state than thoseof other repeat proteins

Both the refolding and unfolding kinetics of TAPLRR can befitted to the sum of two exponential phases (Fig. 2). The ureadependence of the rate constants for these phases, shownin Fig. 3, appears to describe two distinct V-shaped plots.We show that the wild-type data can be modelled reasonablywell using a 3-state mechanism. Most of the mutations affectthe minor refolding/unfolding phase in a similar way to themajor refolding/unfolding phase (Fig. 5 and 6). For example,for L293I and L348I, the major and minor unfolding ratesof the mutant are similar to those of the wild-type; for I264V,the major and minor unfolding rates of the mutant aremuch faster than those of the wild type. One possible reasonfor this could be that the reaction can proceed via twoparallel pathways, one including an intermediate and onegoing directly from D to N. These pathways proceed via similartransition states and are therefore affected by mutations in asimilar way.

There has been some discussion in the literature in recentyears regarding the structural and mechanistic interpretationof F values and to what extent the magnitude of a F value isdependent on the equilibrium stability change associated withthe mutation;32,33 uncertainty may sometimes arise when thestability change is small, and the F value obtained can thenappear to be erroneously high. Therefore, care must be takenwhen carrying out this type of analysis and particularly wheninterpreting the observation of a very small number of high Fvalues amongst otherwise low F values in terms of a nucleation-type folding mechanism. In this regard it is interesting to lookat repeat proteins. For this class of proteins, polarised folding

Fig. 4 Equilibrium denaturation curves of TAPLRR mutants. (A) Mutationsin the N-terminal helical cap, L212V (triangles) and L238I (open circles).(B) Mutations in equivalent positions of repeat 1 (I264V) and repeat 3(L315A) shown by triangles and open circles, respectively. (C) Mutations inequivalent positions of all repeats. L267I in repeat 1 is shown by opendiamonds, L293I and L293V in repeat 2 are shown by crosses and opencircles, respectively. L317I in repeat 3 is shown by triangles. L348I in repeat4 is shown by open squares. The denaturation curve of wild-type TAPLRRis shown by closed circles in all panels for comparison. The data were fittedto a two-state equation as for Fig. 2.

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and unfolding mechanisms have generally been observed todate (see for example ref. 1, 6 and 21), in which structure in thetransition state is localised to a subset of repeats. It is impor-tant to emphasise, however, that this behaviour corresponds to

a large rather than a small number of high F values, and theyare not associated with small stability changes.

A different picture is obtained here for the transition state ofTAPLRR compared with those of previously studied repeat

Fig. 5 Plots of the refolding and unfolding rate constants for TAPLRR mutants. Each panel shows the urea dependence of the rate constants forrefolding and unfolding of wild type (black) and mutant (red). The data are fitted as for Fig. 2. The schematic shows in red the location of each pairof mutants.

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proteins. The transition state is rather diffusely structured, withthree of the four LRRs having residues with high F values ofbetween 0.5 and 1. The difference in the unfolding mechanismof TAPLRR compared with other repeat proteins may be aconsequence of the small number of repeats combined with arelatively large cap, causing TAPLRR to behave more likeglobular proteins that tend to have diffuse transition statestructures. Interestingly, we find that two residues, one locatedin the N-terminal helical cap and one in the first leucine-richrepeat, have anomalous F values of less than 0 or greater than 1,indicating the presence of non-native interactions in the transi-tion state. The formation of non-native structure by the cap maybe required early in the reaction to protect the LRRs while theyfold; the cap must then rearrange to the native structure late inthe folding reaction.

Experimental proceduresMolecular biology and protein purification

TAPLRR (residues 199–372) in plasmid pQE60 (Qiagen) with aC-terminal non-cleavable 6-residue histidine tag was a generousgift from E. Conti (EMBL, Heidelberg, Germany). Site-directedmutagenesis was performed using the QuikChange kit (AgilentTechnologies) and mutant plasmids identified by sequencing.E. coli C41(DE3) cells were transformed with plasmid and asmall number of colonies picked from the plate into flaskscontaining 1 L 2�TY medium plus ampicillin. The cells weregrown at 37 1C to O.D.600nm B 0.6 then induced with 0.5 mMIPTG overnight at 28 1C. Cells were harvested by centrifugationat 5000g for 10 minutes at 4 1C and resuspended in 50 mMTris-HCl buffer, pH 8.1, 500 mM NaCl, 10% glycerol, 5 mMimidazole. Cells were lysed in an Emulsiflex C5 high-pressurehomogeniser (GC Technology) and soluble protein purifiedby Ni-NTA affinity chromatography followed by size-exclusion

chromatography (Superdex 75, GE Healthcare Life Sciences).Purified protein was dialysed into 5 mM Tris-HCl buffer,pH 8.1, and was judged to be 495% pure by SDS-PAGE andmass spectrometry. Protein aliquots were flash frozen andstored at �80 1C.

Equilibrium denaturation

0.8 mL aliquots containing various concentrations of urea inbuffer (50 mM sodium phosphate, pH 7, 1 mM EDTA) weredispensed using a Hamilton MicroLab dispenser. 100 mL pro-tein was added to each aliquot to a final concentration of 4 mMand samples equilibrated at 25 1C for 4 hours. For refoldingexperiments, the protein was first denatured in 3 M urea andincubated for 1 hour at 25 1C, then dispensed as above. Thefluorescence was monitored using an Aminco-Bowman series 2luminescence spectrofluorimeter using a quartz cuvette with a10 mm pathlength. The excitation wavelength was 280 nm andemission was measured between 345 nm and 355 nm at a rateof 1 nm s�1. CD spectra were recorded on a Jasco J720spectrapolarimeter using a quartz cuvette with a 10 mm path-length at 25 1C. Three scans from 240 nm to 217 nm wereacquired and averaged for each urea concentration at a scanrate of 50 nm min�1. The final protein concentration for theCD measurements was 15 mM. Equilibrium denaturationcurves were fitted to a two-state model as described34 usingKaleidagraph 4.1 (Synergy software).

Stopped-flow fluorescence

Stopped-flow fluorescence measurements were performedusing an Applied Photophysics SX-18MV instrument. The finalprotein concentration after mixing was 4 mM. Six traces wereacquired and averaged for each urea concentration. Refoldingexperiments were performed by mixing one volume of proteinunfolded in 3 M urea with 10 volumes of buffer (50 mM sodiumphosphate buffer, pH 7, 1 mM EDTA) at 25 1C. Unfoldingexperiments were performed by mixing one volume of proteinin buffer with 10 volumes of solutions of buffer and urea. Theaveraged traces were fitted to the sum of two exponentialphases using Kaleidagraph 4.1. The V-shaped plots were fittedto a quadratic equation to describe the denaturant-dependentmovement of the transition state according to Hammondbehaviour.

Calculation of U values

F-value analysis is used to characterise the structures of foldingtransition states and intermediates at a residue-specific level.35

By making single-site, conservative mutations that delete aninteraction or a small number of interactions without introdu-cing new interactions, the effect on the energy of the transitionstate can be determined. If a mutation destabilises the transi-tion state by the same amount as it destabilises the native statethen it indicates that that region is as structured in thetransition state as it is in the native state (F = 1). Conversely,if a mutation destabilises the native state but has no effect onthe stability of the transition state then the region can be saidto be as unstructured in the transition state as it is in the

Table 2 Kinetic parameters for the unfolding of wild type and mutants ofTAPLRR

ln kH2Ou mu (M�1) ln k5.5M

u F5.5M

WT �4.18 � 0.03 1.09 � 0.01 0.26 � 0.01L212V �2.74 � 0.12 0.99 � 0.02 1.13 � 0.03 0.63 � 0.13L238I �2.08 � 0.12 1.02 � 0.02 1.93 � 0.03 o0I264V �2.48 � 0.07 0.99 � 0.01 1.39 � 0.02 �0.05 � 0.17L267I �4.07 � 0.09 0.95 � 0.02 �0.42 � 0.03 41L293I �3.65 � 0.11 1.01 � 0.02 0.33 � 0.03 0.95 � 0.04L293V �4.18 � 0.10 1.05 � 0.02 0.01 � 0.03 1.11 � 0.03L315A �2.74 � 0.09 1.05 � 0.02 1.47 � 0.02 0.57 � 0.07L317I �2.57 � 0.93 0.94 � 0.02 1.04 � 0.03 0.46 � 0.09L348I �4.04 � 0.06 1.07 � 0.01 0.29 � 0.02 0.97 � 0.04

ln kH2Ou and mH2O

u were obtained by fitting the unfolding data to aquadratic equation. To minimise the errors, a weighted-average value ofthe curvature was calculated using the wild type and mutants (hm*i =�0.052). The data were then refitted to a quadratic equation with m*fixed to the weighted-averaged value. The data were also fitted to givethe rate constants for unfolding at 5.5 M urea, in the middle of thedenaturant concentration range used for the unfolding kinetics, inorder to avoid the need for large extrapolation back to 0 M urea andthereby reduce the errors. The errors listed are the standard deviationsfrom the fits of the data. The F values were calculated at 5.5 M urea andthey have errors of 5–10%.

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Fig. 6 Plots of the rate constants of the minor phase of refolding and unfolding for TAPLRR mutants. Each panel shows the urea dependence of the rateconstants for wild type (black) and mutant (red). The schematic shows in red the location of each pair of mutants.

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6458 | Phys. Chem. Chem. Phys., 2014, 16, 6448--6459 This journal is© the Owner Societies 2014

denatured state (F = 0). Fractional values are most simplyinterpreted as resulting from partial structure formation inthe transition state. F values were calculated for TAPLRR fromthe unfolding rate constants using the following equation:

F ¼ 1�RT ln

kmutu

kwtu

� �

DDGD�N

where T is the temperature in Kelvin, R is the gas constant, andkwt

u and kmutu are the rate constants for unfolding of wild-type

and mutant proteins, respectively. DDGD–N is the change in thefree energy of unfolding upon mutation as measured by equili-brium denaturation experiments.

Modelling

The data were fitted using the following equations, derivedin ref. 30:

l1;2 ¼1

2b1 �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib12 � 4b2

p� �

with

b1 = k1 + k2 + k3 + k4 + k5 + k6

b2 = k1(k3 + k4 + k6) + k2(k4 + k5 + k6) + k3(k5 + k6) + k4k5

where l1,2 are the two experimental rates and the ki are themicroscopic rates as defined in Fig. 3. The on- and off-pathwaymechanisms can be obtained from the above general expres-sion by setting the relevant rates to zero. In addition to theseequation an exponential dependence of the ki on the denaturantconcentration was assumed:

ki = kH2O exp(mi[U])

where kH2O is the rate without denaturant and [U] is thedenaturant concentration.

The amplitudes for the triangular mechanism are given by:

Aunfolding ¼k4l1 � g3 þ a k6l1 � g2ð Þð Þ

l1 l1 � l2ð Þ

Arefolding ¼k3l1 � g1 þ ð1� aÞ k2l1 � g2ð Þð Þ

l1 l1 � l2ð Þ

with

g1 = k5k3 + k5k2 + k1k3

g2 = k6k3 + k6k2 + k4k2

g3 = k6k1 + k4k5 + k4k1

a ¼ FI � FD þ mI �mDð Þ½U�FN � FD þ mN �mDð Þ½U�

where FN, FU, FI, mN, mU, mI are the fluorescence intensities andtheir change with urea of the native, and denatured states(which can be obtained from the equilibrium denaturation)and of the intermediate. For further details see ref. 29 and 30.

We moreover added Hammond behaviour in the eventualfits of the major phase, i.e. the dependence of the rates on ureawas changed to:

ln ku½U� ¼ ln kH2Ou þmu½U� þm�½U�2

We found this was necessary as a 3-state model withoutHammond behaviour is not capable of fitting both phases andstill produce the correct curvature for the major phase. Theresulting system, a 3-state reaction with Hammond behaviour,is rather complex, but was the simplest mechanism consistentwith the observed data.

Acknowledgements

This work was supported by the Medical Research Council of theUK (grant G1002329) and the Medical Research Foundation. Wethank Dr Markus Seeliger for carrying out preliminary biophysicalexperiments and Dr Alan Lowe for helpful discussions regardingdata analysis. GM was supported by the Cambridge Home and EUScholarship Scheme.

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