Protein Folding Cooperativity and Thermodynamic Barriers of the

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Protein Folding Cooperativity and Thermodynamic Barriers of the Simplest #-Sheet Fold: A Survey on WW Domains Manuel Iglesias-Bexiga, Malwina Szczepaniak, Celia Sanchez-Medina, Eva S Cobos, Raquel Godoy-Ruiz, Jose CRISTOBAL Martinez, Victor Munoz, and Irene Luque J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b05198 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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The Journal of Physical Chemistry

Protein Folding Cooperativity and Thermodynamic Barriers of the Simplest β-Sheet Fold: A Survey on WW Domains Manuel Iglesias-Bexiga†, Malwina Szczepaniak‡, Celia Sánchez de Medina‡, Eva S. Cobos†, Raquel Godoy-Ruiz§, Jose C. Martinez†, Victor Muñoz‡,¶,*and Irene Luque†,* †

Department of Physical Chemistry and Institute of Biotechnology. University of Granada, Granada, Spain ‡ Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Darwin 3, 28049 Madrid, Spain § Department of Chemistry & Biochemistry, University of Maryland, College Park, MD 20742, USA ¶ Department of Bioengineering, University of California Merced, Merced, CA 95343, USA *

Authors for correspondence: I.L. phone: +34 958 240440; email: [email protected]; V.M. phone +1 209 2282430; email [email protected]

Running title. Folding cooperativity in WW domains

Abbreviations: NEDD4, human Neuronal precursor cell Expressed Developmentally Downregulated 4 ubiquitine ligase; YAP65, human Yes Associated Protein; FBP11, human Formin Binding Protein 11; DSC, Differential Scanning Calorimetry; CD, Circular Dichroism; FT-IR, Fourier Transform-Infrared Spectroscopy; N, native state; U, unfolded state; Mw, molecular weight; Tm, temperature at mid-denaturation; ΔHm, enthalpy of unfolding at Tm; ∆GN-U(T), Unfolding Gibbs energy; ∆HN-U(T), unfolding enthalpy; ∆SN-U(T), unfolding entropy; ∆CpN-U(T), unfolding heat capacity

1 ACS Paragon Plus Environment

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ABSTRACT

Theory and experiments have shown that microsecond folding proteins exhibit characteristic thermodynamic properties that reflect the limited cooperativity of folding over marginal barriers (downhill folding).

Those studies have mostly focused on

proteins with large α–helical contents and small size, which tend to be the fastest folders. A key open question is whether such properties are also present in the fastest all-beta proteins. We address this issue investigating the unfolding thermodynamics of a collection of WW domains as representatives of the simplest β–sheet fold. WW domains are small microsecond folders, although they do not fold as fast as their α– helical counterparts. In previous work on the NEDD4-WW4 domain, we reported deviations from two-state thermodynamics that were less apparent, and thus suggestive of an incipient downhill scenario. Here we investigate the unfolding thermodynamics of four other WW domains (NEDD4-WW3, YAP65-WW1(L30K), FBP11-WW1 and FBP11-WW2) by performing all of the thermodynamic tests for downhill folding that have been previously developed on α–helical proteins. This set of five WW domains shares low sequence identity and include examples from two specificity classes, thus providing a comprehensive survey. Thermodynamic analysis of the four new WW domains consistently reveals all of the properties of downhill folding equilibria, which are in all cases more marked than what we found before in NEDD4-WW4. Our results show that fast-folding all-beta proteins do share the limited cooperativity and gradual unfolding thermodynamics with fast α-helical proteins, and suggest that the free energy barrier to folding of natural proteins is mostly determined by size and fold topology, and much less by the specific amino-acid sequence.


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INTRODUCTION WW domains are very small (35 to 50 amino acids) and versatile polyproline recognition domains that form a stable antiparallel three-stranded β-sheet structure, the simplest fold within the all-beta protein class. Because of their small size, fast folding kinetics and resilience against mutation, these domains have been intensively and extensively used as models to investigate fast folding kinetics, both experimental and computational (see1 for a review). WW domains are in fact the fastest folding natural all-beta proteins identified to date2, with folding times ranging from 20 to 100 microseconds. Most recently, the WW fold has been the focus of studies aimed at pushing the folding speed limit of all-beta proteins using protein engineering and rational design 3. Microsecond folding kinetics and the limited unfolding cooperativity that is often detected in high-resolution equilibrium experiments of fast folding proteins are both tightly connected to the downhill folding scenario4. Small size is also a strong predictor of downhill folding5. Those properties, together with their elementary antiparallel topology, make WW domains ideal candidates for exploring the downhill folding mechanism1,6. In fact, accumulating evidence from kinetic and equilibrium studies suggests that WW domains often fold by crossing small free energy barriers. Conformational equilibrium studies of YAP and FBP28 WW domains show apparent two-state folding behavior that, in the case of FBP28-WW, can be turned into a threestate mechanism by effects of temperature, truncation or mutation7-10. Computational studies with Pin1-WW led to similar conclusions, suggesting that this domain exhibits downhill behavior below physiological temperatures11,12. Additionally, a detailed thermodynamic study of the fourth WW domain in human NEDD4 led some of us to propose that the conformational equilibrium of this domain is at the frontier between



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cooperative folding and a one-state continuous downhill equilibrium13. Consistently with the latter, a recent computational study of the folding pathways of several fast folding domains has identified NEDD4-WW4 as a downhill folder 14. One of the key questions that emerges from previous work in fast folding proteins in general, and WW domains in particular, is to what extent is the downhill folding character built into the structural properties of the fold versus how much it depends on the specific amino acid sequence. The best way to address this question is by performing a thorough comparative analysis of structural homologues.

Such

comparison has been performed previously between two examples of ultrafast folding helix-loop-helix domain: BBL, a paradigm of one-state downhill folding15, and PDD, a BBL-homologue that folds slower and with more cooperativity16. The comparison between equilibrium and kinetic properties nicely demonstrated the correlation between thermodynamic unfolding cooperativity and folding speed17. Here we address the same question from the viewpoint of beta-proteins, and using WW domains, which fold onto the simplest beta fold, as model systems. An additional advantage is that there are many naturally occurring WW domains, offering the possibility of comparing structural homologues that vary vastly in sequence due to divergent evolution. In previous work13, we showed that the conformational equilibrium of the fourth WW domain of the human ubiquitin ligase NEDD4 (NEDD4-WW4) exhibits the thermodynamic properties indicative of an incipient downhill folding scenario in which the folding free energy barrier is present but fairly small, and the native structure is flexible. These properties include: steep baselines in conventional equilibrium unfolding experiments, large heat capacities for the native state and very low values for the specific enthalpy and temperature of maximal stability16,18,19. Using NEDD4-WW4 as our reference, in this work we perform a survey study in which we have characterized


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the unfolding cooperativity and the thermodynamic folding barrier of four additional domains, containing two representatives of each of the two major WW classes: i) Class I WW domains, which recognize peptide sequences containing a PPxY consensus motif (domains NEDD4-WW3 and YAP65-WW1(L30K) as well as the previously studied NEDD4-WW4); and ii) Class II WW domains, which interact with PPLP containing sequences (domains FBP11-WW1 and FBP11-WW2). These domains fold into the same native structure and have sequence homology below 30% (see Figure 1), and, thus, are as divergent as possible for proteins sharing the same native structure20.

MATERIALS AND METHODS Protein samples Genes encoding for the sequences of the third WW domain of human NEDD4, NEDD4-WW3, the L30K mutant of the first WW domain of human YAP65, YAP65WW1(L30K), and the two WW domains of human FBP11, FBP11-WW1 and FBP11WW2, corresponding to the amino acids 834-878 (UniProtKB/Swiss-Prot code P46934), 165-207 (UniProtKB/Swiss-Prot code P46937), 134-177 and 175-219 (UniProtKB/Swiss-Prot code O75400), of their respective full length proteins, were cloned into the pETM30 expression vector (EMBL Core Purification Facility) as previously described 13. All protein domains were expressed in E. coli BL21/DE3 cells as N-terminal GST-His-tag fusion proteins with a TEV protease restriction site engineered between the WW domain and the His-tag and purified as described before

13

. Additionally, the

samples were further purified using a second step of nickel affinity chromatography followed by a gel filtration step on a HiLoad Superdex 75 column (GE healthcare Life Science). The purity and integrity of the proteins were checked by SDS-Page and Mass



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Spectrometry (Mass Spectrometry Service of the CIC at the University of Granada) and estimated to be above 99%. Immediately after the purification, all samples were frozen in liquid nitrogen in the purification buffer (50 mM sodium phosphate buffer, 300 mM, NaCl pH 8.0) and stored at -80 ºC. Under these conditions proteins are stable for several months. Samples for experimental work were prepared by extensive dialysis against a large volume (1-2 L) of the appropriate buffer at 4 ºC: 20 mM sodium phosphate at pH 7.0, 20mM MES at pH 6.0, 20mM sodium acetate either at pH 5.0 and pH 4.0, 20 mM glycine either at pH 3.5 and pH 3.0, and 20 mM phosphoric acid buffer at pH 2.0. Urea solutions were prepared as described elsewhere 13. Protein concentration was measured by absorption spectroscopy at 280 nm using the following molecular weights and extinction coefficients (determined according to Gill and von Hippel21): 5568 Da and 11380 M-1·cm-1 for Nedd4-WW3, 5720 Da and 11660 M-1·cm-1 for Nedd4-WW4, 5539 Da and 12600 M-1·cm-1 for YAP65WW1(L30K), 5323 Da and 15440 M-1·cm-1 for FBP11-WW1 and 5697 Da and 16990 M-1·cm-1 for FBP11-WW2.

Circular

Dichroism,

Fluorescence

and

Fourier

Transformed

Infrared

Spectroscopy Measurements Circular dichroism (CD) measurements were performed with a Jasco J-715 spectropolarimeter and tryptophan fluorescence measurements were performed with an Eclipse spectrofluorimeter (Cary Varian) as described in13. Both instruments were equipped with a temperature controlled cell holder. The Fourier Transformed Infrared (FT-IR) experiments were recorded in an Excalibur FTS-3000 Spectrometer (BioRad) as is described in17.


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The spectroscopic (CD, fluorescence and FT-IR) thermal denaturation profiles were fitted to the two-state model as described before 13,17. For the individual fittings of the experimental traces the Tm (the denaturation temperature where the N and U populations are equal to 50%), ΔHm (the denaturation enthalpy value, referenced at Tm), and the values of the y-intercepts and slopes of the linear functions describing the baselines for both N and U states were considered as floating parameters. In the initial stages of the analysis, ΔCpN-U was taken to be zero. The set of thermal unfolding curves obtained at different pH values, or at different urea concentrations, were fitted using the same equations considering different Tm and ΔHm values for each curve and common baselines for the N and U states. In these cases ΔCpN-U was obtained from the slope of the ΔHm vs Tm regression by an iterative process, setting initially ΔCpN-U to zero and iterating the analysis until no changes in the unfolding heat capacity were obtained between two successive rounds. The Gibbs energy [∆GN-U(T)], enthalpy [∆HN-U(T)], entropy [∆SN-U(T)] and heat capacity [∆CpN-U(T)] functions for the denaturation process were subsequently derived from the van´t Hoff and Kirchhoff relationships.

Differential Scanning Calorimetry DSC experiments were conducted in a VP-DSC instrument (Microcal Inc.) and individually fitted to a two-state model (N⇄U) as described in

13

. For the global

analysis of the DSC traces at different pH values, common heat capacity functions for the N and U states at all pH conditions were considered 22, and individual values for the Tm and ΔHm parameters were derived for each pH. The pH dependence of the unfolded state heat capacity associated to changes in the protonation state of charged amino acids



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was taken into account in the definition of the unfolding heat capacity according to the equation:

∆C pN −U = C pU − C pN + Fp ·∆C pPROT

(1)

where CpN and CpU are the native and unfolded heat capacity functions, defined as linear and parabolic functions respectively 13, ΔCp,PROT(100 ºC) is the maximum contribution to the heat capacity due to side-chain protonation and Fp is the degree of protonation. ΔCp,PROT(100 ºC) is considered as a fixed parameter directly measured from the DSC profiles as the difference between the Cp,U(100 ºC) values at pH 3 and 7, while Fp is defined as a floating parameter ranging from 0 to 1 for each pH condition. Analysis of the DSC profiles at pH 7.0 according to the variable-barrier model was performed using to the equations described in 23. Taking into account the molecular weight of each domain, we have proceeded to the individual fitting of the respective DSC traces. The model has four adjustable parameters, which determine the general properties of the folding ensemble: i) the “characteristic” temperature, T0; ii) the height of the barrier, β; iii) the difference in enthalpy between the minima found at low and high temperature, ∑α, where for the two-state scenario it will be a good estimate of the transition enthalpy at T0; iv) an asymmetry factor, f, where if f=1 the heat capacity is the same for both minima at T0 and if f>0, two macrostates) or as a barrierless transition (β≤0, downhill) by simple visual inspection.

RESULTS Sequence, structural and spectroscopic features of WW domains The five WW domains investigated in this study were cloned with the exact same length so that the influence of the N- and C-terminal regions, known to play a role on the stabilization of these domains, was as uniform as possible. From an amino acid sequence standpoint, the five domains are very different, exhibiting highly divergent sequences with some pairs sharing less than 30% of their sequence (Figure 1A). The domains were also selected because they all have small loops of the same size, thus minimizing any possible impact of loop length on the folding regime. This is particularly relevant for loop 1, connecting strands β1 and β2, which is proposed to be the rate-limiting step for folding of Pin1-WW on the basis of the effects that changes in its length had on the folding rate24,25. Despite their interest as folding models, high resolution structural information on WW domains is scarce. However, the crystal structures of YAP65-WW1(L30K) (4REX26) and NEDD4-WW3 (4N7H27) and the NMR structure of the FBP11-WW1 (1ZR728) domain are available, which permit a direct comparison. As illustrated in Figures 1B and S1, these three domains fold into essentially the same native 3D structure, as demonstrated by their very low rmsd values: 9 ACS Paragon Plus Environment


0.6 Å between the two x-ray structures and of 1.2 Å between those and the FBP11WW1 NMR structure. The WW fold is characterized by a twisted three-stranded β-sheet stabilized by a “hydrophobic buckle” formed at the core of the domain, which implicates a highly conserved Trp residue in strand β1, an aromatic residue (generally Phe or Tyr) in strand β3, and two conserved Pro residues at the N- and C- termini. In all three available structures, the four residues forming this hydrophobic buckle occur in the same overall conformation. Importantly, the network of hydrophobic contacts stabilizing the native fold is also highly conserved among the three domain structures. The only potential exceptions are some interactions implicating residues at the Ntermini that are missing in the NMR structure of FBP11-WW1. Due to the high content in aromatic residues of their minicore, WW domains generally present far-UV CD spectra with a characteristic positive absorption band centered at 220-230 nm associated to the formation of the hydrophobic buckle that plays an essential structural role in the stabilization of the domain29,30. An additional strongly negative peak at 200 nm is also observed, characteristic of short and irregular β strands7,8,13,31,32. The Far-UV CD spectrum of WW domains is thus sensitive to the integrity of the native core and of the native sheet hydrogen bonding. Figure 1C shows the CD spectra for the five WW domains, all exhibiting these two diagnostic features. There are, however, distinct differences in the shape and intensity of the two bands between the five proteins. These differences do not correlate with their class ascription (i.e. whether class 1 or class 2). For instance, the spectra at 2 ºC and neutral pH (highest native stability) show marked changes in intensity of the positive 220-230 nm band: very sharp and intense for FBP11-WW2; slightly less intense for FBP11-WW1 and NEDD4-WW4; broader and weaker for YAP65-WW1(L30K); and almost non-existing for NEDD4-WW3. It is also noteworthy that the negative band corresponding to the


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natively distorted β sheet shifts its position rather drastically, ranging from 205 nm for NEDD4-WW3 to 195 nm for FBP11-WW2. These differences in far-UV CD spectra are indicative of a certain degree of variability among the five domains in the dynamics of the long-range contacts defining the β-sheet structure and in the degree of packing of aromatic residues.

Thermal unfolding experiments consistently reveal probe-dependent behavior The equilibrium thermal unfolding of the five WW domains was measured probing the process with various spectroscopic techniques that report on different structural aspects of the protein, including fluorescence spectroscopy (hydrophobic packing in tertiary structure), FTIR spectroscopy (secondary structure) and Far-UV CD (combination of secondary structure and hydrophobic packing for WW domains). The thermal unfolding of the five WW domains was highly reversible (over 90% in all cases) and no appreciable scan rate or concentration effects were observed, indicating that the five domains are monomeric in the protein concentration and temperature range of our experiments, and obey a standard folding-unfolding equilibrium. The upper panels in Figures S2-S4 show the far-UV CD, fluorescence and FTIR spectra for the different WW domains at temperatures ranging from 2 to 98 ºC. The lower panels display the resulting thermal unfolding profiles (using a single wavelength in each case, and normalized to facilitate comparison). In general, we observe that the thermal unfolding transitions measured with the three techniques are sigmoidal but rather broad. Moreover, the pre- and post-transition baselines for the three probes on the five WW domains show strong temperature dependence that is suggestive of folding near the downhill scenario 33.



The normalized unfolding curves of the five WW domains were fitted one by one to a two-state model, which reproduces the individual data well (Figure 2). However, the resulting thermodynamic parameters (Tm and ∆Hm) obtained from each probe on each domain differ significantly (Table 1). For instance, the Tm values obtained by Trp fluorescence (reporting on its tertiary environment) are systematically lower than those resulting from the analysis of the changes in the amide-I band using FTIR spectroscopy, which reports on the loss of secondary structure (antiparallel βsheet hydrogen bonding in this case). The differences range from 4 ºC for NEDD4WW3 to 8 ºC for FBP11-WW1. As discussed in the previous section, the far-UV CD spectrum of WW domains has contributions from both secondary structure and the tertiary environment of the aromatic residues, and their relative contributions vary depending on the WW domain. Accordingly, the far-UV CD unfolding behavior is more similar to either the FTIR or the fluorescence behavior depending on which spectral contribution is stronger for that particular WW domain. In domains characterized by intense and well-defined positive bands at 230 nm (i.e. strong contributions from tertiary contacts associated to the hydrophobic buckle such as NEDD4-WW4 and FBP11-WW2) we find Tm values from CD that are very close to those derived from Trp fluorescence experiments (Table 1). Conversely, domains characterized by smaller contributions from aromatic residues to the CD spectrum (YAP65-WW1(L30K) and NEDD4-WW3) show Tm values in these experiments that are similar to those obtained by FTIR. In this regard, the CD behavior of FBP11-WW2 constitutes an exception since it presents an intense band at 230 nm, but its unfolding curve is closer to the FTIR curve, as it occurs for YAP65-WW1(L30K) and NEDD4-WW3. The differences in Tm between probes observed for each domain are highly significant, and point to the marginal unfolding cooperativity expected for the downhill scenario33. Probe


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dependence is consistently observed for all of the WW domains. Particularly, they all show a higher Tm (by an average of 6 ±1.5 ºC) for the melting of secondary structure than for melting the aromatic tertiary environment. That is, these proteins loose their tertiary structure earlier (in terms of a temperature scale) than their secondary structure, exactly as it has been previously documented for the α–helical one-state downhill folder BBL34 and for the α+β fast folder gpW 35. The various spectroscopic probes also produce differences in the apparent ∆Hm. Here the patterns are less obvious, possibly because in contrast to the Tm, the ∆Hm obtained from a two-state fit is highly interdependent with the native and unfolded baselines33. Nevertheless, we see that, in general, the far-UV CD curves tend to give higher ∆Hm and the FTIR curves lower ∆Hm, whereas fluorescence curves exhibit somewhat intermediate values. The variability amongst probes for each protein is rather high (46-50 kJ·mol-1). Comparing proteins, we find that NEDD4-WW3, YAP65WW1(L30K) and FPB11-WW2 feature smaller ∆Hm than NEDD4-WW4 and FBP11WW1. In qualitative terms, the data suggest that NEDD4-WW4 experiences the most cooperative unfolding of the five domains, whereas NEDD4-WW3 and YAP65WW1(L30K) are the least cooperative. The divergence in Tm and ∆Hm values determined from the various spectroscopic probes allow us to rule out a two-state scenario and are indicative of the limited cooperativity expected for downhill folding36. Even though the parameters obtained from the two-state analysis are not thermodynamically meaningful since these proteins do not adhere to this regime, they are, nonetheless, useful as diagnostic indicators of marginal unfolding cooperativity and of the decoupling that exists in these domains between the melting of tertiary and secondary structure.



Differential scanning calorimetry indicates that WW domains have partially disordered native states and unfold with limited cooperativity The thermal unfolding of the five WW domains was also studied by differential scanning calorimetry (DSC). The calorimetric profiles were recorded over a wide pH range (from 7 to 3) in order to optimize baseline definition (Figure 3). Under all tested conditions, a high level of reversibility was found in all cases with the exception of FBP11-WW2, which at pH 7.0 and 5.0 was found to be irreversible, even in the presence of reducing agents. Nonetheless, when scanning was stopped at temperatures just above completion of the main unfolding transition (around 70-80 ºC) 95% reversibility was obtained. Those results indicated that up to such temperatures the unfolding process of FBP11-WW2 is at near equilibrium, permitting a complete thermodynamic analysis. Thus, for this protein at pH values between 7.0 and 5.0, we analyzed the DSC data only up to 80 ºC. The DSC profiles of the WW domains displayed extremely broad transitions with poorly defined baselines, showing a progressive increment of the unfolded state heat capacity with pH. The only exception are the aforementioned FPB11-WW2 traces in the 7.0-5.0 pH range, which showed anomalously low Cp,U values, due to posttransition aggregation processes occurring at high temperature. In all other cases (YAP65-WW1(L30K), NEDD4-WW3 and FBP11-WW1), the Cp,U(100ºC) values at pH 3.0 are close to the theoretical estimates derived from the tabulated contributions of individual amino acids 37, whereas at pH 7.0 they are about 2 kJ·K-1·mol-1 higher than predicted. The pH dependence of Cp,U(T) has been previously described and attributed to the different ionization states of charged amino acid side-chains contributing differently to the overall heat capacity value

38

. Because the magnitude of these

contributions is small and close to the detection limit of most DSC instruments, they are


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typically ignored for most proteins. Nonetheless, these effects become significant for small proteins such as our WW domains, which show small and broad unfolding peaks in DSC experiments. The groups susceptible to change their ionization state in the different WW domains and an estimate of their contribution to the Cp,U are summarized in Table S1. Overall, the changes in Cp,U(T) with pH can be well accounted for as arising from these contributions. To better define Cp,N(T) and Cp,U(T), and thus obtain a realistic description of the folding equilibrium for each WW domain, the DSC curves at different pH values were globally fitted to a two state model considering common Cp,N(T) and Cp,U(T) functions, as described before13, but including, in this case, a linear pH dependence for Cp,U(T) (see Methods). The results of this analysis are summarized in Table 2. As shown in Figure 3, the two-state model reproduces the global DSC as a function of pH data for each WW domain, with the exception of NEDD4-WW3 for which the global two-state analysis failed to converge (we show the individual fits at each pH instead). However, in all cases, the global two-state analysis renders physically implausible baselines that intersect at temperatures close to the Tm that are far from the universal convergence temperature normally found for globular two-state proteins (i.e. ~120 ºC) 37. Moreover, the Cp,N(T) functions obtained from the fits do not agree with the predictions from the Freire analysis based on the protein molecular weight

39

, which works very well for

two-state globular proteins. Instead, here we find Cp,N(T) functions that are 2-2.5 kJ·K1

·mol-1 higher than the theoretical estimates and have much steeper temperature

dependence. These features are indicators of conformational heterogeneity of the native state and/or of the gradual structural disordering (uncooperative unfolding) that is characteristic of the downhill folding scenario34,40.



In our previous analysis of NEDD4-WW4 we could derive a more physically plausible Cp,N(T) function from a global two-state analysis that included the CD thermal unfolding curves at different pH values together with the DSC data13. Such analysis rested in three assumptions: i) DSC thermograms share common Cp,N(T) and Cp,U(T) functions; ii) CD curves share native and unfolded state baselines; and iii) the ∆Hm and Tm values are the same for DSC and CD at each pH condition. We attempted the same analysis using DSC and CD data as a function of temperature and pH for all other WW domains. We could only achieve convergence for YAP65-WW1(L30K), for which the two-state model provided a reasonable fit with Cp,N(T) and Cp,U(T) functions that crossed at a temperature near 100 ºC (somewhat closer to the convergence temperature). For the remaining WW domains this fitting strategy failed, indicating that the DSC and CD unfolding data are disparate, as we observed before for CD, fluorescence and FTIR (see above). Such differences are illustrated in Figure 4, where we compare the CD temperature versus pH experiments for each WW domain with the corresponding CD profiles simulated using the thermodynamic parameters derived from the analysis of the DSC data. In summary, DSC analysis confirms the conclusions derived from the multiprobe spectroscopic study, namely that the conformational equilibria of all of the WW domains studied here do not conform to the two-state paradigm. In general, the twostate model cannot account for the experimental data globally and, when it is used to interpret individual datasets (i.e. of single probes or at a single pH value), it renders implausible thermodynamic parameters (summarized in Table 2) that differ from those previously observed on globular proteins that unfold via a two-state, highly cooperative process. Specifically, the two-state fits estimate an unfolding enthalpy for WW domains in a range between 40 and 45 J·g-1 at 110 ºC, which is well below the standard value for


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globular proteins (54 J·g-1)

41

. This is strongly indicative of marginal unfolding

cooperativity. This anomalously low unfolding enthalpies are accompanied by large native-state heat capacity values (Cp,N(20 °C)) that exceed the upper limit expected for globular proteins (1.25-1.8 J·K-1·g-1

42

), and by values for the temperature of maximal

stability (Ts) that are unrealistically low. Both of these observations indicate that the native state experiences a high level of conformational flexibility even in conditions at which is the most stable species. For instance, at neutral pH, where the native-state stability is highest, the estimated Ts for the WW domains ranges between -34 °C and -9 °C, constituting, to the best of our knowledge, the lowest values ever described for globular proteins 43,44.

Double perturbation experiments confirm the minimally cooperative unfolding of the WW fold Another experimental test of compliance of an equilibrium unfolding transition with a barrier-limited (two-state) process is based on the coupling between two different unfolding procedures such as thermal and chemical denaturation45. In these experiments, a linear change of the thermal denaturation parameters as a function of the concentration of denaturant indicates adherence to a barrier-limited transition. In contrast, a gradual unfolding (or barrier-less in thermodynamic terms) process in which the conformational ensemble changes continuously should result in non-linear dependence of the thermal unfolding parameters on the denaturant concentration45. We thus performed double perturbation experiments on NEDD4-WW3, YAP65WW1(L30K), FBP11-WW1 and FBP11-WW2 combining thermal and urea denaturation to complement those we previously performed on NEDD4-WW413. In these experiments, it is very important that the spectroscopic probe used to monitor



unfolding is sensitive to the local conformation (secondary structure) of the protein, which is the structural property most likely to reveal non-cooperative unfolding behavior

45

. We used far-UV CD as probe because FTIR experiments cannot be

accurately performed in the presence of high chemical denaturant concentrations, in spite the fact that the CD spectrum of WW domains has mixed contributions from secondary and tertiary structure (see above). Figure 5 shows the thermal denaturation curves of the WW domains monitoring the native structure by CD at 230 nm and obtained in the presence of increasing urea concentrations (0 to 6 M). The figure also shows the best global fit to the two-state model (left panels). A gradual downshift of the native CD signal (i.e. at low temperature) occurs as the urea concentration increases for NEDD4-WW3, YAP65-WW1(L30K) and FBP11-WW2. We previously found the same behavior on NEDD4-WW4 13, in which case the observation could still be explained as the onset of cold denaturation within a two-state scenario because the global fit of the temperature-urea data to a two-state model rendered: a) non-intersecting native and unfolded baselines; b) ∆Cp,N-U value consistent with that obtained from simple thermal denaturation; and c) ∆Hm and Tm values that decreased linearly with increasing urea concentration. Although the global fit for NEDD4-WW4 was seemingly consistent with two-state unfolding, the anomalous values for the thermodynamic parameters (∆Hm, Ts and Cp,N(20 °C)) obtained by DSC led us to propose that this domain unfolds as an incipient downhill folder in which the thermodynamic free energy barrier separating the native and unfolded macrostates is small and comparable to thermal energy 13. For NEDD4-WW3, YAP65-WW1(L30K) and FBP11-WW2, we find that the two state model can also reproduce the experimental data (left panels in Figure 5), resulting in ∆Hm and Tm values that show reasonably good linear correlations with urea concentration and ∆Cp,N-U values that agree with those derived from the two-state DSC 18 ACS Paragon Plus Environment

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analysis (Tables 2 and 3). However, the changes in ∆HN-U(25 °C) as a function of urea concentration obtained from the fits (right panels in Figure 5) do not obey the Maxwell relationships expected for a two state equilibrium transition17,35,45. Particularly, the fits impose an ∆HN-U(25 °C) that increases linearly with addition of urea, a change that is not physically plausible. Therefore, we conclude that the double perturbation experiments on NEED4-WW4, NEDD4-WW3, YAP65-WW1(L30K) and FBP11WW2 are strongly indicative of unfolding processes with limited cooperativity, as expected for low thermodynamic free energy barriers. The behavior of FBP11-WW1 in the double perturbation experiments is the one that most directly points to a marginally cooperative unfolding process. For FBP11-WW1 the global two-state fit was unable to account for the experimental data using a common ∆Cp,N-U for all the experiments (various urea concentrations), and the individual fits rendered Tm and ∆Hm values that changed in the notoriously non-linear ways (Figure 5) that correspond to equilibrium unfolding processes within the downhill scenario.

Extracting thermodynamic folding barriers of WW domains from DSC The differential scanning calorimetry thermogram of protein unfolding is directly related to the molecular partition function. This relationship implies that it is possible to derive the properties of the conformational ensemble of the protein from an analysis of the DSC experiment, including the population of the highest free energy partially folded species separating the native from the unfolded sub-ensembles (i.e. the barrier top)23,46. We employed the original variable barrier model to extract the thermodynamic folding barrier for all the WW domains from their DSC thermograms obtained under conditions of maximal stability (pH 7) 23. This model fits the DSC data to a 1D-free energy surface defined as a quartic Landau polynomial as a function of the 19 ACS Paragon Plus Environment


unfolding enthalpy as the folding order parameter23. The analysis renders four fitting parameters that define the folding free energy surface of the protein under study and its thermodynamic transition: T0 (or temperature at which the native and unfolded minima have the same free energy); ∑α, which defines the difference in enthalpy between the native and unfolded minima; f which determines the asymmetry between the width of the native and unfolded minima; and β, which is the height of the thermodynamic folding free energy barrier. The analysis performed on the DSC thermograms in absolute heat capacity units of the five WW domains (at pH 7) rendered fits of high quality despite the fact that is uses fewer fitting parameters than a comparable two-state fit (i.e. 4 vs. 6; upper panel of Figure 6). The parameters from the fits are given in Table 4. As it can be seen in Figure 6 (lower panel), the folding free energy surface at T0 of the five WW domains shows two broad minima corresponding to partially (dis)ordered native and unfolded ensembles separated by a small to minuscule free energy barrier, or even a single broad minimum near the center. Of the five domains, only FBP11-WW2 displays an appreciable barrier between the unfolded and native state. However, this barrier (β = 1.4 kJ·mol-1) is still smaller than thermal energy, and thus it results in high populations at the characteristic temperature of the species conforming the barrier (Figure S5). We can conclude that FBP11-WW2 folds within the general downhill folding scenario11. FBP11-WW1 and NEDD4-WW4 present tiny barriers (β values below 0.2 kJ·mol-1) that result in populations of the species at the barrier top that are only slightly lower than those of the ground states (native and unfolded) (Figure S5). For YAP65-WW1(L30K) and NEDD4-WW3 no thermodynamic free energy barrier is observed, and thus the population at the characteristic temperature shows a maximum corresponding to the partly unfolded conformations equivalent to those that conform the barrier top in the other WW domains. Therefore, according to the variable barrier model 20 ACS Paragon Plus Environment

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analysis of the DSC experiments, these WW domains unfold following a global downhill or one-state unfolding process 4,11.

DISCUSSION The detailed thermodynamic analysis of fast-folding proteins unveils deviations from two-state behavior that result from their limited unfolding cooperativity4. Kinetically, these proteins are expected to fold downhill because they approach the folding speed limit2, which has been consistently estimated to be microseconds by multiple methods, including the timescales of elementary processes, such as secondary structure formation47 and hydrophobic collapse48, size-scaling effects on folding rates5, or estimates of the mean folding transition path time49. Thermodynamically, the marginal cooperativity of the downhill folding scenario can be detected using various purposely developed tests: 1) observation of broad unfolding curves in which the native state becomes gradually disordered, as manifested by skewed native and unfolded baselines and/or a high, positively sloped, native heat capacity33,36; 2) observation of non-concerted

unfolding

when

the

process

is

monitored

by

different

structural/spectroscopic probes15; 3) non-linear coupling between the effects caused by two denaturing agents45; 4) a broad DSC thermogram that when analyzed in terms of the entire folding/unfolding conformational ensemble leads to significant populations of the highest free energy, partially folded conformations in the ensemble (i.e. the thermodynamic barrier top)23,46. All these tests were developed using the one-state downhill folder BBL as proof of concept, and each one of them has been subsequently applied to other fast-folders4. However, most of those proteins have native folds that are either fully α–helical, such as BBL15,34, PDD16,17, mutants of λ–repressor50, the P22 subdomain51 and the de novo designed αtα52, or largely α–helical such as gpW35. In



fact, the only fast-folding, all-beta protein that has been looked at in this context is NEDD4-WW4, which only showed subtle deviations from two-state behavior13. An important unaddressed question was then whether all-beta fast folding proteins exhibit as little unfolding cooperativity in equilibrium as fast folders with α–helical or mixed folds. Here we directly address this question by performing a comprehensive survey of the unfolding thermodynamics of four other WW domains. We find that the five representatives of the WW fold exhibit complex, gradual unfolding equilibria that are incompatible with two-state (or barrier-limited) folding, and, instead, indicative of the downhill scenario. The thermodynamic signatures of downhill folding are consistently found in the four tests outlined above for any given WW domain, and also across all of the domains investigated in this study. We can thus unambiguously conclude that fastfolding all-beta proteins share the limited cooperativity and gradual unfolding thermodynamics previously documented on largely α–helical fast-folders. The significance of this conclusion is two-fold. First, it indicates that the connection between fast (i.e. near the speed limit) folding kinetics and minimally cooperative equilibrium unfolding transitions is universal, and thus independent of the protein structural class. Second, it highlights how careful quantitative analysis of protein unfolding thermodynamics can provide key insights about folding mechanisms that are lost in the conventional two-state analysis. The limited cooperativity of downhill folding proteins makes them ideal models to dissect the molecular determinants and evolutionary constraints that define the free energy barriers to folding1,4. In this regard, our survey on the unfolding thermodynamics of WW domains, and their comparison with similar data on other downhill folders, offer key generalizations. For instance, WW domains fold in microseconds, but are still slower than fast-folders composed of α–helices1,6. Therefore, the discovery that their


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unfolding thermodynamics are downhill-like necessarily indicates that the folding speed limit for all-beta folds must be lower than that for α–helical or mixed folds, thus confirming theoretical predictions derived from polymer scaling arguments53. The five WW domains studied here have little sequence identity (>30%), yet they all share, to varying extents, the thermodynamics of downhill folders. This implies that structural properties of the native WW fold, such as size and antiparallel arrangement, have much stronger influence on the folding free energy barrier and cooperativity than do any changes in energetics arising from their differences in amino acid sequence. Interestingly, multiprobe unfolding experiments unveil a highly conserved trend by which the denaturation midpoint for probes that report on tertiary structure (e.g. fluorescence) is systematically lower than for probes that report solely on secondary structure (e.g. amide-I FTIR) (see Table 1). This observation indicates that tertiary contacts defining the fold of these domains are lost earlier (in terms of a temperature scale) than are the networks of hydrogen bonds conforming the β-sheet secondary structure. Incidentally, this exact same pattern has been observed by NMR in the atomby-atom analysis of BBL34 and gpW 54, in multiprobe unfolding experiments on PDD16, on site-specific unfolding experiments on P22 subdomain51 and a de novo designed αtα domain52, and even on NMR unfolding experiments in the slow conformationalexchange regime of the two-state folder α–spectrin SH3 domain55. The confluent pattern of tertiary structure melting at lower temperatures than secondary structure leads us to conclude that this is a universal property of protein folding, even though it may be difficult to detect on very slow two-state folders. The thermodynamic features of downhill folding are apparent in all of the five WW domains. Nonetheless, there is some variability in spectroscopic and thermodynamic unfolding behaviors that is ultimately associated to their distinct amino 23 ACS Paragon Plus Environment


acid sequences. In this way, NEDD4-WW3 domain has the strongest signatures of downhill folding, whereas the next domain in the multi-domain protein NEDD4 (i.e. NEDD4-WW4) presents the subtlest signatures, which required detailed quantitative analysis to effectively rule out a two-state scenario

13

. The other three domains show

downhill signatures at intermediate degrees between these two extremes. The thermodynamic folding barriers estimated from DSC also differ slightly (Figures 6 and S5). However, all such differences are so small compared to thermal energy that it may be unrealistic to expect strong correlations between the behaviors derived from the different criteria. There are, however, some overall trends worth discussing. We find an inverse correlation between the native heat capacity (i.e. Cp,N(20 °C)) and the relative intensity of the CD signal at 230 nm (Figure 7A). The Cp,N(20 °C) reflects the magnitude of the enthalpy fluctuations of the native ensemble38,39 and the positive CD band at 230 nm reports on the packing of aromatic residues31. Consequently, their inverse correlation points to these properties being similarly sensitive to the structural flexibility inherent to the native WW fold. Moreover, excluding the clearly off-trend, very high native heat capacity value for NEDD4-WW3, which is probably due to this domain being marginally stable resulting in partial unfolding at room temperature, the native heat capacity inversely correlates with the barrier height (β) derived from DSC (Figure 7A; r2 of 0.93). A good linear correlation is also found between β and the CD ellipticity at 230 nm (Figure 7B; r2 = 0.84), indicating that the folding cooperativity of these domains is connected to specific spectroscopic features seen in equilibrium experiments. This correlation suggests that the integrity and strength of the characteristic hydrophobic buckle of the WW fold

13

is a structural determinant of

cooperative folding for WW domains. The residues implicated in the buckle, and their interactions, are all highly conserved among the five WW domains (see Figure S1).


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Therefore, the variability observed in thermodynamic unfolding behavior is likely to arise from interactions with other residues that surround the hydrophobic buckle and modulate its stability. The high-resolution crystal structure of YAP65-WW1(L30K) revealed the formation of a well-organized cluster of salt-bridges and water-mediated hydrogen bonds that implicate an Arg residue at the back of the beta sheet and four other charged residues at the N- and C- termini of the domain26. Such cluster is thought to be responsible for the strong pH dependence of the stability of the native structure26. A similar pH dependence was observed for all the WW domains studied herein. Unfortunately, the N- and C- terminal ends are not well defined in the available NMR structures of the NEDD and FBP11 WW domains, which impedes us to assess whether equivalent interactions are also found in the other domains. But in any event, several reports exist in the literature indicating that the length of the WW domain construct has strong effects on defining whether the isolated domain is folded or not 56,57.

CONCLUSIONS Our detailed thermodynamic analysis of the unfolding process of WW domains demonstrates that the simplest β–sheet fold domains experience high conformational flexibility in their native state, as it is inherent to the downhill folding scenario. A conformationally heterogeneous native state could have functional significance by allowing promiscuous binding as well as efficient modulation of binding affinity and specificity with relatively minor changes in conditions4,58,59. Notably, these conclusions are in agreement with recent results suggesting that the recognition of peptide ligands by WW domains might take place through coupled folding/binding equilibria, so that both, direct interactions at the binding site and the conformational dynamics of the domain, play key roles in determining the binding energetics


60,61

. In this regard,


NEDD4-WW3, which we find it here to be partly disordered in native conditions by virtue of its marginally stable downhill folding equilibrium, has been identified before as the main player in establishing the NEDD4 protein-protein interaction network. This confluence is noteworthy because NEDD4 contains other three WW domains (including the WW4 studied here) with highly similar sequences (over 65%). Therefore, it seems that additional factors other than structural complementarity must be important in determining binding affinity and specificity of these WW domains62. In the light of our results, and considering the hypothesis that folding barriers might act as modulators of conformational plasticity and binding promiscuity4,59, it is enticing to hypothesize that the differences in cooperativity between the one-state downhill NEDD4-WW3 and the almost barrier-limited NEDD4-WW4 are significant determinants of their different behavior towards protein recognition.


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ASSOCIATED CONTENT Supporting Information Protonation heat-capacity changes, crystal structures of WW domains, equilibrium thermal unfolding experiments, probability densities obtained from the variable-barrier model (PDF)

AUTHOR INFORMATION Corresponding Authors (I.L.) E-mail: [email protected]; phone: +34 958 240440 (V.M.) E-mail [email protected]; phone +1 209 2282430 ORCID Eva S. Cobos: 0000-0002-4622-8698 Jose C. Martinez: 0000-0003-2657-2456 Irene Luque: 0000-0003-2757-4779 Victor Muñoz: 0000-0002-5683-1482 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The work was funded by grants BIO2012-39922-CO2 from the Spanish Ministry of Science and Innovation and BIO2016-78746-C2-1-R from the Spanish Ministry of Economy (to I. L.), and ERC-2012-ADG-323059 from the European Research Council (to V.M.), as well as by the Fondo Europeo de Desarrollo Regional (FEDER). V.M. also acknowledges support from the W.M. Keck foundation and the National Science Foundation

through

grants

NSF-CREST-1547848

(Center


for

Cellular

and


Biomomolecular Machines) and NSF-MCB-1616759. Mass spectrometry measurements were performed at the Center of Scientific Instrumentation of the University of Granada.

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Page 32 of 45

Table 1. Two-state analysis of the equilibrium data for the five WW domains at pH 7 a. CD

Fluorescence

FT-IR b

DSC c

Tm (oC) FBP11-WW1

66.0 ± 1.7

65.3 ± 1.1

73.4

67.0 ± 1.6

FBP11-WW2

59.0 ± 1.4

55.0 ± 1.3

59.8

53.1 ± 3.9

NEDD4-WW3

55.8 ± 3.9

50.8 ± 2.5

55.1

58.3 ± 3.7

NEDD4-WW4

57.2 ± 0.6

57.3 ± 0.7

64.1

58.1 ± 1.5

YAP65WW1(L30K)

56.8 ± 2.1

50.1 ± 1.1

56.1

46.9 ± 0.8

∆Hm (kJ·mol-1) FBP11-WW1

119 ± 10

95 ± 8

73 ± 8

127 ± 8

FBP11-WW2

105 ± 10

104 ± 12

77 ± 15

110 ± 6

NEDD4-WW3

92 ± 22

98 ± 22

62 ± 5

92 ± 5

NEDD4-WW4

134 ± 7

109 ± 7

83 ± 10

129 ± 8

YAP65WW1(L30K)

93 ± 12

96 ± 5

85 ± 14

a

94 ± 5

The 95% confidence level of the fit parameter is given. Tm values of FTIR were fixed to the respective values obtained from 1D-FES analysis of the IR T-jump data c These data have been collected from individual fitting of DSC traces to the two-state model b


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Table 2. Thermodynamic parameters of WW domains obtained from two-state global analysis of DSC thermal unfolding experiments under neutral and acidic pH conditions a. pH Tm Cp,N ∆Hm ∆GN-U ∆Cp,m ∆h,m TS ∆Gmax(TS) (oC) (kJ·mol-1) (25oC) (oC) (kJ·mol-1) (kJ·K1·mol-1) (110oC) (20oC) (kJ·mol-1) (J·g-1) (J·K-1·g-1) FBP11WW1 b

FBP11WW2 c

NEDD4WW3 d

NEDD4WW4 e

YAP65WW1

Fp

7

67.9

129

17.4

-34

32.5

41.0

5 4 3 7

69.2 66.3 55.3 57.0

111 107 100 120

15.7 14.0 9.4 8.8

-31 -30 -25 -26

27.9 25.2 18.8 16.0

40.0 37.0 36.8 41.0

1.73

0.78 0.36 0 N.A.

5 4 3 7

57.7 52.9 34.6 58.8

111 102 93 83

5.6 3.1 -1.2 8.7

-19 -18 -15 -9

10.6 8.6 3.5 13.3

38.1 40.6 39.7 42.5

1.70

N.A. N.A. N.A. N.A.

6 5 4 3.5 7

50.4 48.3 37.7 30.3 58.0

79 63 54 52 133

5.1 3.7 1.9 0.9 11.6

6.7 4.3 2.9 2.5 19.5

6 5

46.5 37.4

100 73

5.9 2.6

4

22.6

41

-0.3

7 5

48.5 45.6

101 91

5.8 5.4

5 11 12 9 25.0 -16.0 9.0 3.0 -24 -23

11.0 6.0

1.02

1.89

2.1

2.5

41.2 39.9 39.6 36.0

45.0

1

2.05

1.73

1.8 11.4 10.9


N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A.

2.0

43.9 46.5

1.72

1 0.52

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

(L30K) b

4 3.5

33.6 21.8

73 57

2.5 -0.6

-20 -17

7.2 3.7

a

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44.2 42.5

0 0

Errors have been estimated as 5% for Tm, 10% for ∆Hm and 20% for the rest of parameters. The heat capacity functions derived from fitting have been modulated by the protonation degree, Fp, according to the equation: ∆CpN2 U = CpU(T) – CpN(T) + Fp·∆Cp,prot, where CpN(T) = 9.16 + 0.05·(T-293), CpU(T) = 9.98 + 0.0224(T-293) - 0.0001(T-293) and o -1 -1 ∆Cp,prot(100 C) = 1.4 kJ·K ·mol for FBP11-WW1; CpN(T) = 9.50 + 0.03·(T-293), CpU(T) = 10.90 + 0.0224·(T-293) - 0.0001·(T293)2 and ∆Cp,prot(100oC) = 1.0 kJ·K-1·mol-1 for YAP65-WW1(L30K). The slope coefficients and the quadratic terms in the CpU(T) function were estimated from the individual contributions of amino acids and chemical groups 37 (see Materials and Methods for details). c The protonation degree could not be estimated from the experimental data, so we used CpN(T) = 9.46 + 0.05·(T-293), CpU(T) = 9.80 + 0.0239·(T-293) – 0.0001·(T-293)2 and ∆Cp,prot(100oC) = 0 kJ·K-1·mol-1. d Shown are the results from the individual fittings: CpN(T) = 11.17 + 0.07·(T-293) and CpU(T) = 12.50 + 0.0181·(T-293) – 0.0001·(T293)2 at pH 7; CpN(T) = 10.68 + 0.05·(T-293) and CpU(T) = 12.52 + 0.0181·(T-293) – 0.0001·(T-293)2 at pH 6; CpN(T) = 10.14 + 0.08·(T-293) and CpU(T) = 12.43 + 0.0181·(T-293) – 0.0001·(T-293)2 at pH 5; CpN(T) = 10.25 + 0.08·(T-293) and CpU(T) = 12.26 + 0.0181·(T-293) – 0.0001·(T-293)2 at pH 4; CpN(T) = 10.12 + 0.08·(T-293) and CpU(T) = 11.94 + 0.0181·(T-293) – 0.0001·(T-293)2 at pH 3.5. e The results for NEDD4-WW4 have been extracted from 13. b


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Table 3. Thermodynamic parameters of WW domains obtained from two-state analysis of CD double-perturbation experiments at pH 7

FBP11-WW1 FBP11-WW2 NEDD4-WW3 YAP65-WW1(L30K)

Tm (oC) 64.2 51.8 51.5 48.9

∆Hm (kJ·mol-1) 112 128 134 132

∆GN-U(25oC) (kJ·mol-1) 10.0 8.2 7.7 7.8


∆Cp,m (kJ·K1·mol-1) 1.24 2.10 2.90 2.20


Table 4. Fitting results of the DSC traces of the five WW domains at pH 7 by using the variable-barrier model 23 ∑α To R·To β f (kJ·mol(K) (kJ·mol-1) (kJ·mol-1) 1 ) FBP11-WW1 111.14 340.66 2.8 0.1150 0.8973 FBP11-WW2 141.25 328.26 2.7 1.3997 1.0000 NEDD4-WW3 228.11 330.51 2.7 -1.0000 0.9822 NEDD4-WW4 127.68 332.09 2.7 0.1628 0.8404 YAP65-WW1 226.48 320.24 2.6 -1.2934 0.5808 (L30K)


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Figure 1. (A) Sequence alignment of the five WW domains under study. Identities and similarities are shaded in the same color than the residues. The N-terminal motif GAMG results from the cloning procedure. Residues participating in the hydrophobic buckle are marked by arrows (B) Ribbon diagram of a NEDD4-WW3 domain. Side chains of binding site residues and residues organizing the conserved hydrophobic buckle are shown as sticks. (C) Far-UV CD spectra of the five WW domains.



Figure 2. Multi-probe equilibrium thermal unfolding profiles for the five WW domains. Symbols correspond to the normalized spectral data as a function of temperature for fluorescence spectroscopy (green), Far UV-CD (black) and FT-IR (orange). Solid lines in the same color code represent their individual fits to the two-state model. The midpoint of each transition (native probability of 0.5) is marked by vertical dashed lines.


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Figure 3. Temperature dependency of the partial molar heat capacity for the five WW domains. Symbols correspond to the normalized heat capacity profiles at pH 7.0 (brown), 5.0 (orange), 4.0 (light green) and 3.0 (dark green). The best fit to a two-state model is shown as solid black lines. The results for NEDD4-WW4 domain have been extracted from 13.



Figure 4. Temperature dependence of the ellipticity signal at 230 nm for the five WW domains. Symbols correspond to the experimental profiles recorded at pH 7.0 (brown), 5.0 (orange), 4.0 (light green) and 3.0 (dark green). The expected denaturation profiles according to the two-state DSC analysis as shown as solid black lines. The native and the unfolded states baselines are shown as discontinuous black lines. The results for NEDD4-WW4 domain have been extracted from 13.


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Figure 5. Left panels: Urea dependence of the far-UV CD thermal denaturation profiles for the five WW domains. Experimental data points are shown as small circles. Urea concentrations are 0 M (brown), 0.5 M (orange), 1.0 M (light green), 2.0 M (dark green), 3.0 M (cyan), 4.0 M (blue) purple 5.0 M (purple). For FBP11-WW1 three additional urea concentrations were included: 6.0 M (brown), 7.0 M (orange), 8.0 M (black). The results of the global fitting to a two-state model are shown as black lines. The native and unfolded state baselines obtained in the fits are shown as dashed lines. Right panels: Unfolding enthalpy values at 25 oC vs urea concentration. The linear regression of data is shown as a grey dashed line. The results corresponding to NEDD4-WW4 domain have been extracted from 13.


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Figure 6. Upper panel: DSC thermal unfolding transitions of the five WW domains at pH 7 (circles) and the best fit (solid lines) to the variable-barrier model, using the native-state baseline estimated from molecular weight (straight lines below the experimental traces) 39. Lower panel: the Gibbs energy profiles at the characteristic temperature, T0.



Figure 7. Correlation between the barrier height, β, estimated from the variable-barrier model analysis and (A) the heat capacity of the native state measured at 20oC or (B) the molar ellipticity maximum at 230 nm measured by CD, for the five WW domains under study. Linear regressions of the data are shown as solid black lines.


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TOC Figure


Protein Folding Cooperativity and Thermodynamic Barriers of the

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