Cooperative Interactions and a Non-native Buried Trp in the Unfolded State of an SH3 Domain

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1 doi: /s (02) available online at on Bw J. Mol. Biol. (2002) 322, Cooperative Interactions and a Non-native Buried Trp in the Unfolded State of an SH3 Domain Karin A. Crowhurst 1,2, Martin Tollinger 1 and Julie D. Forman-Kay 1,2 * 1 Department of Structural Biology and Biochemistry The Hospital for Sick Children 555 University Avenue Toronto, Ont. Canada M5G 1X8 2 Department of Biochemistry University of Toronto, Toronto Ont., Canada M5S 1A8 *Corresponding author The presence of residual structure in the unfolded state of the N-terminal SH3 domain of Drosophila drk (drkn SH3 domain) has been investigated using far- and near-uv circular dichroism (CD), fluorescence, and NMR spectroscopy. The unfolded (U exch ) state of the drkn SH3 domain is significantly populated and exists in equilibrium with the folded (F exch ) state under non-denaturing conditions near physiological ph. Denaturation experiments have been performed on the drkn SH3 domain in order to monitor the change in ellipticity, fluorescence intensity, and chemical shift between the U exch state and chemically or thermally denatured states. Differences between the unfolded and chemically or thermally denatured states highlight specific areas of residual structure in the unfolded state that are cooperatively disrupted upon denaturation. Results provide evidence for cooperative interactions in the unfolded state involving residues of the central b-sheet, particularly the b4 strand. Denaturation as well as hydrogen-exchange experiments demonstrate a non-native burial of the Trp ring within this cooperative core of the unfolded state. These findings support the presence of non-native hydrophobic clusters, organised by Trp rings, within disordered states. q 2002 Elsevier Science Ltd. All rights reserved Keywords: unfolded state; SH3 domain; cooperativity; residual structure; NMR Introduction Structural characterisation of disordered states is vital for comprehensive understanding of the mechanism of protein folding. Preferential sampling of native-like conformations may initiate folding or non-native structure may lead to kinetic traps. In addition, recent evidence has shown that disordered states serve functional roles in vivo in disorder-to-order transitions during protein recognition, in some cases providing plasticity to enable multiple binding partners. 1 Disordered states are implicated directly in a variety of cellular processes, including interaction with chaperones, Abbreviations used: drkn SH3 domain, the N-terminal SH3 domain of Drosophila drk; U exch and F exch, the unfolded and the folded state of the drkn SH3 domain; U Gdn, the denatured state of the drkn SH3 domain in $2.0 M guanidinium chloride; U temp, the denatured state of the drkn SH3 domain at $70 8C; Gdn, guanidinium chloride; HSQC, heteronuclear single quantum coherence; SAXS, small-angle X-ray scattering; DSS, 2,2- dimethyl-2-sila-pentane-5-sulfonate; FID, free induction decay. address of the corresponding author: forman@sickkids.on.ca protein translocation across membranes and vesicle fusion mediated by SNARE proteins, which are intrinsically unstructured in isolation but which assemble into a complex through binding-induced folding. 2,3 Unfolded and partially folded proteins also play crucial roles in a number of disease states such as amyloidoses and cancer. 1 Disordered states are ensembles of rapidly interconverting conformations. 4 These ensembles are difficult to characterise because experimental information reflects an average of the characteristics of the structures within the ensemble. In general, disordered states are quite distinct from random coil; under some conditions they can be highly compact and show evidence for large amounts of residual secondary structure, which may resemble or differ from the folded state. 5 7 Many of the advances in understanding disordered states have relied on NMR, the most powerful tool for studying the details of ensembles of exchanging conformations. 8 For example, a combination of experiments including nuclear Overhauser effect (NOE) measurements, paramagnetic relaxation enhancement as well as chemical shift analysis has provided evidence for substantial native-like residual structure in D131D, a partially folded /02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

2 164 Interactions and Buried Trp in Unfolded SH3 Domain Figure 1. Ribbon diagram of the folded state (A. U. Singer et al., unpublished results) and sequence of the drkn SH3 domain. The locations of the seven b-strands in the folded structure are indicated by arrows. The Figure was generated using MOLSCRIPT. 55 fragment of staphylococcal nuclease, stabilised primarily by local and medium-range interactions. 9,10 The molten globule of a-lactalbumin (at low ph) and its mutants show marked resistance to denaturation in the core of the protein, as well as the retention of native-like secondary structure and topology in the a-domain, while the b-domain is fairly unstructured. 11 Mutagenesis experiments on protein L have indicated the formation of one b-hairpin before another in the folding transition state and paramagnetic relaxation enhancement experiments are consistent with the stabilisation of this first b-hairpin in the denatured state (in 2 M guanidinium chloride (Gdn)). 7,12 One unfolded protein state that has been characterised extensively is the N-terminal SH3 domain of the Drosophila protein drk (drkn SH3 domain, Figure 1), a homologue of the vertebrate Grb2 protein. When isolated, the drkn SH3 domain exists in slow exchange on the NMR chemical shift timescale between folded (F exch ) and significantly populated unfolded (U exch ) states under non-denaturing aqueous buffer conditions. 13 In many studies characterising disordered states, unfolding is induced by harsh conditions of temperature, ph, chemical denaturant or by mutagenesis. The unfolded state of the drkn SH3 domain, however, can be studied in the absence of denaturant or physical modifications to the protein. This environment is representative of a more physiologically relevant disordered state, and it permits direct comparison to the folded state under the same conditions, as well as to denatured states. It is important to clarify our nomenclature in this regard, with the unfolded (U exch ) state referring to the ensemble of structures of the drkn SH3 domain that exists in equilibrium with the folded (F exch ) state under non-denaturing conditions (50 mm sodium phosphate buffer, ph 6 at 5 8C) and denatured states representing ensembles of conformations that exist under conditions of chemical or thermal denaturation, i.e. the U Gdn denatured state in $2.0 M Gdn and the U temp denatured state at $ 70 8C. A large body of previous work has provided evidence for residual structure in the U exch state of the drkn SH3 domain. The 1 H 15 N heteronuclear single quantum coherence (HSQC) spectrum shows peak-broadening for residues 23 28, caused by intermediate exchange with a low population of specific stabilised conformations. 15 J HNHA coupling experiments indicate non-native a-helical propensity for residues in the U exch state. 15 The U exch state contains compact conformations having transiently populated secondary structure and tertiary contacts, whereas chemically and thermally denatured states are closer to random coil, having more extended conformations and few measurable intramolecular interactions. This has been demonstrated using NMR diffusion and relaxation experiments as well as small-angle X-ray scattering (SAXS), with the measured hydrodynamic radius and radius of gyration, respectively, of the unfolded state more compact than expected from a random coil structure. 17 Sidechain relaxation experiments exhibit restriction of motion for aromatic rings, in particular the single Trp36 ring. 18 NOE and fluorescence experiments also highlight the Trp36 indole group, which appears more buried in the U exch state than the folded state, where it is located on the surface. 16 Much of the previous characterisation of the U exch state has focussed on comparison to the folded state of the drkn SH3 domain. Here, our primary approach is to compare the U exch and denatured states. Differences between them imply that the U exch ensemble contains a greater degree of compact structure than the denatured state. A series of denaturation experiments have been performed, utilising a variety of spectroscopic methods including NMR, far-uv and near-uv CD and fluorescence, in order to compare the structural characteristics of the U exch state to that of thermally or chemically denatured states. In addition, hydrogen-exchange rates of backbone amide and Trp indole protons have been determined to further probe the stability of the residual structure in the drkn SH3 domain U exch state. Results Standard unfolding experiments map the transition from a folded state of a protein to a denatured (or unfolded) state, with the use of chemical or thermal denaturants. Our experiments differ from those traditional studies, since the

3 Interactions and Buried Trp in Unfolded SH3 Domain 165 transition to be monitored is between the U exch and the denatured (U Gdn or U temp ) states. As described previously, 13 under non-denaturing conditions, the drkn SH3 domain can be found in equilibrium between a folded (F exch ) and a significantly populated unfolded (U exch ) state. Because this conformational exchange is slow on an NMR chemical shift timescale, a proton nitrogen correlation spectrum (HSQC) shows twice the expected number of peaks: half corresponding to the F exch state and the other half to the U exch state. It is due to the separation of the two states that NMR is such a powerful tool for studying the U exch state. An additional advantage of NMR over other techniques is the residue-specific or site-specific data enabling fine structural detail to be obtained. When a denaturant is added to the sample, the F exch peaks disappear and the U exch peaks shift, reflecting a change in the populations of the conformations within the U exch ensemble. The titration is complete when the peaks no longer shift. This new spectrum represents an ensemble of conformations with few persistent interactions and more extended structure that corresponds to the denatured state (U Gdn or U temp ). In monitoring the chemical shift change for each residue from the U exch to the denatured state, we can monitor how specific areas of the molecule in the unfolded state are structurally affected by denaturation. Fluorescence and CD techniques do not permit the separation of the F exch and U exch contributions, and the data therefore represent an average of the two states. One approach to deconvoluting the two contributions is to compare the F exch /U exch denaturation data to experiments performed on the fully folded state (F s ) of the drkn SH3 domain, stabilised by the addition of 0.4 M Na 2 SO 4. Since chemical shifts for residues in the F exch and F s states are nearly identical, 13 it is likely that the folded states have the same structure. By comparing data from a fully folded and the composite F exch /U exch states of the drkn SH3 domain, it is possible to infer the contribution of the U exch state alone. Figure 2. Maximal observed 15 N chemical shift change for U exch state residues as a function of residue position (a) between 0 M and 2.7 M Gdn at 5 8C and (b) between 5 8C and 70 8C. The last datum point represents N 11 of Trp36. NMR guanidine denaturation experiment Gdn was titrated from 0 M to 2.7 M into a sample of 1.3 mm 15 N-labelled drkn SH3 domain in the F exch /U exch states (50 mm sodium phosphate, ph 6). Upon addition of Gdn, F exch peaks in the 1 H 15 N HSQC spectra disappear quickly (no F exch peak is seen at 0.6 M), while the U exch state peaks move. Their final positions represent the spectrum for the guanidine-denatured state of the drkn SH3 domain, U Gdn. For HSQC spectra of both the F exch / U exch equilibrium and the U Gdn states, see Zhang et al. 13 Amide nitrogen chemical shifts were used as reporters of structural changes during the transition between the U exch and U Gdn states. The 15 overall N chemical shift change from 0 M to 2.7 M Gdn as a function of residue (Figure 2(a)) shows that some regions of the protein undergo much greater changes in electronic and electrostatic environment than others. Note, in particular, residues 17 29, a region of the molecule where evidence for residual structure has been observed previously. 15 The data can be analysed by plotting a normalised amide 15 N chemical shift change as a function of Gdn concentration for each residue in the protein (Figure 3). Most residues display curves ranging from simple power functions to exponentials (Figure 3(b)). However, for a small subset of residues, the data are best fit to a sigmoidal curve, with transition points ranging from 0.7 M to 1.2 M Gdn (Figure 3(a)). Note that some of these residues correspond to peaks that have moved significantly over the course of the titration. The Hill equation (see equation (1) in Materials and Methods) can be used to provide a numerical estimate, called the Hill coefficient (H ), of the degree of sigmoidal curve shape. Values near 1 indicate no sigmoidal character, while increasing

4 166 Interactions and Buried Trp in Unfolded SH3 Domain Figure 3. (a) and (b) 15 N chemical shift change from U exch to U Gdn states (normalised) as a function of [Gdn]. (c) and (d) 15 N chemical shift change from U exch to U temp states (normalised) as a function of temperature. (a) and (c) Representative residues whose curves can be fit to a sigmoidal function. (b) and (d) A representation of curve shapes for all other residues. values above 1 denote increasingly steep sigmoidal transitions. Table 1 summarises the Hill coefficients for all residues. Data in boldface represent residues with H $ 1.6. Most missing entries indicate that the total chemical shift change is too small to achieve a good fit for the Hill equation; in some cases, however, curves cannot be fit due to excessive scatter in the data arising from difficulties in precise measurement of chemical shifts for highly overlapped peaks. NMR thermal denaturation experiment A thermal titration from 5 8C to708c was also performed on a 0.5 mm drkn SH3 domain sample in 50 mm sodium phosphate (ph 6), 50 mm 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The thermal denaturation is fully reversible at this concentration of protein. The F exch peaks in the 1 H 15 N HSQC spectra disappear (at about 50 8C, not as quickly as in the Gdn titration), and the U exch peaks again undergo a change in chemical shift. The unfolded resonances at the end of the thermal denaturation are representative of the U temp state, and the chemical shifts do not correspond to those observed at the end of the Gdn experiment. This U temp state is therefore distinct from both the U exch and U Gdn states. In plotting the overall 15 N chemical shift change as a function of residue for this data (Figure 2(b)), the maximal chemical shift change was measured between the 15 N chemical shift at 5 8C and the highest temperature at which the resonance is still visible, since in a number of cases the peak disappears or broadens out at a temperature below 70 8C. There is, as in the Gdn titration, an aboveaverage chemical shift change for residues Note that the shift is in the direction opposite to that seen for the same residues in the Gdn experiment. While the total average chemical shift change is larger for the thermal denaturation, this may be due to unresolved issues in referencing

5 Interactions and Buried Trp in Unfolded SH3 Domain 167 Table 1. Summary of Hill coefficients derived from curve-fitting NMR titration data Residue H Gdn a R 2 Gdn b H temp a R 2 temp b E2 A ^ I ^ A ^ ^ K ^ ^ H ^ D ^ ^ F ^ 0.39 c ^ S ^ ^ A ^ ^ T ^ ^ A ^ ^ D ^ ^ D ^ E ^ ^ L ^ S ^ ^ F ^ R ^ ^ K ^ T ^ ^ Q ^ ^ I ^ ^ L ^ ^ K ^ ^ I ^ ^ L ^ ^ N ^ ^ M ^ E ^ D ^ D ^ ^ S ^ ^ N ^ W ^ W36 side-chain 1.35 ^ Y ^ R ^ ^ A ^ E40 L41 D ^ ^ G ^ ^ K44 E ^ G ^ L ^ ^ I ^ ^ S ^ N ^ Y ^ ^ I ^ ^ E ^ ^ M ^ ^ K ^ N ^ ^ H ^ ^ D ^ a The Hill coefficient for Gdn and thermal denaturation data, respectively. b The coefficient of determination (quality of regression fit) for Gdn and thermal denaturation data, respectively. c Boldface values indicate residues with significant sigmoidal curve shape (H. 1.6). (see Materials and Methods). Interestingly, the resonances of His58, His7 and Asp8 weaken and disappear much earlier than any other residues in the U exch state (with the exception of the extreme Figure 4. Gdn denaturation experiments monitored by tryptophan fluorescence at 5 8C. F exch /U exch data (W) recorded at 358 nm, F s data (X) recorded at 369 nm. N-terminal residues). In particular, the amide peaks for residues 7 and 8 become severely weakened between 20 8C and 30 8C, and all three peaks disappear completely by 45 8C. When assessing the dependence of 15 N chemical shift change on temperature, as in the Gdn titration, a small number of curves are best fit to a sigmoidal shape (Figure 3(c)), with t m values ranging from 31 8C to428c and Hill coefficients of $1.6 (Table 1). These residues either correspond directly to, or are in the same region as, those having sigmoidal transitions in the Gdn experiment. NMR data from chemical and thermal denaturations could not be fit to thermodynamic equations due to the absence of significant baselines required for this type of regression analysis (Figure 3(a) and (c)). Equilibrium fluorescence experiments Fluorescence was used as a spectroscopic probe to monitor the change in environment of the single Trp36 upon Gdn denaturation, starting from both the F exch /U exch equilibrium and F s states (Figure 4). Under equilibrium conditions (F exch /U exch ), chemical denaturation of the drkn SH3 domain does not produce a sigmoidal curve. Assuming a twostate denaturation, the F s -derived sigmoidal curve was fit to a thermodynamic equation (equation (2) in Materials and Methods), from which the DG H2O (unfolding free energy in 0.4 M Na 2 SO 4 ) and m-value (measure of increased exposure of hydrophobic residues upon denaturation) were obtained (Table 2). No data are presented for thermal denaturation of the drkn SH3 domain monitored by Trp36 fluorescence, since intensity changes are masked by thermally induced quenching and there is almost no l max shift during the experiment (about 3 nm total), as the tryptophan residue in the drkn SH3 domain is partially exposed in both the folded and unfolded states.

6 168 Interactions and Buried Trp in Unfolded SH3 Domain Table 2. Summary of Hill coefficients and thermodynamic parameters derived from curve-fitting CD and fluorescence data A. Trp fluorescence: Gdn denaturation C m, Hilla (M Gdn) b H Gdn R 2 Hill c DG H2Od (kcal mol 21 ) m e R 2 therm c F exch /U exch 1.08 ^ F s 1.77 ^ ^ ^ ^ B. Far-UV CD: thermal denaturation t m, Hillf (8C) b H temp R 2 Hill DH mg (kcal mol 21 ) t m, thermf (8C) R 2 therm F exch /U exch 38.4 ^ ^ ^ ^ F s 59.6 ^ ^ ^ ^ C. Near-UV CD: Gdn denaturation monitoring Trp l C m, Hill (M Gdn) H Gdn R 2 Hill DG H2O (kcal mol 21 ) m R 2 therm F exch /U exch 0.46 ^ ^ ^ ^ a Median transition concentration of Gdn. b Hill coefficient for Gdn and thermal denaturation data, respectively. c Coefficient of determination (quality of regression fit) for the Hill and thermodynamic equations, respectively. d Free energy of unfolding or denaturation in water. e The m-value: a measure of the protein s sensitivity to denaturant. f Median temperature of denaturation, calculated by Hill and thermodynamics equations, respectively. g Enthalpy change at t m. Equilibrium circular dichroism experiments Thermal denaturation of both the F exch /U exch and F s states yield sigmoidal transitions reflecting loss of secondary structure when monitored by far-uv CD, although the t m (melting temperature) for the F exch /U exch states occurs at a lower temperature than that of the F s state (Figure 5). A wavelength of 205 nm was chosen to monitor the thermal melts because maximum differences in CD signal were seen at this wavelength, but melting curves at other wavelengths, including 215 nm, the characteristic b-sheet band, also show sigmoidal transitions. The Hill equation was applied to baseline-corrected F exch /U exch and F s data, since plots with non-horizontal baselines have overestimated Hill coefficient values. The t m and DH m values were derived (Table 2). Figure 5. Thermal denaturation of the F exch /U exch state (W) and the F s state (X) monitored by far-uv CD; mean residue weight ellipticity at 205 nm as a function of temperature. No far-uv CD results of the Gdn denaturation were obtained, since the maximum changes in ellipticity from both F exch /U exch and F s states to U Gdn were not significant. Near-UV CD wavelength scans were performed on the F exch /U exch,f s and U Gdn states of the drkn SH3 domain at 5 8C (Figure 6(a)), to probe the asymmetric environments around and interactions between aromatic side-chains, with more intense peaks observed for more rigid side-chains. 19 While the F exch /U exch spectrum represents two sets of conformational ensembles, due to the additive nature of CD spectra, this can be deconvoluted. At 5 8C and ph 6, NMR spectra show that the ratio of folded to unfolded states is approximately 55:45. Since there is strong NMR evidence that the F s and F exch state structures are the same, subtraction of 55% of the F s spectrum from the F exch /U exch spectrum should yield a spectrum representing the U exch state. The three spectra for the F s,f exch / U exch and calculated U exch states all show evidence for peaks arising from the two phenylalanine residues (with sharp, fine peaks between 255 nm and 270 nm), the two tyrosine residues (with one peak between 275 nm and 282 nm), and the single tryptophan residue (whose primary peak is at approximately 290 nm). Chemical denaturation of the F exch /U exch state monitored by near-uv CD at this tryptophan peak wavelength (290.6 nm) yields a sigmoidal transition (Figure 6(b)) whose calculated parameters are listed in Table 2. Reliable results could not be obtained for the F s state, possibly due to aggregation of the protein caused by high concentrations of protein and salt. Thermal denaturations monitored by near- UV CD for both states (F exch /U exch and F s ) were unsuccessful due to very small total changes in ellipticity.

7 Interactions and Buried Trp in Unfolded SH3 Domain 169 Figure 7. Protection factors from solvent exchange of backbone and Trp indole NH groups in the U exch state as a function of residue number. Backbone NH protection factors are shown as black bars, while the lower limit for the tryptophan N 11 H protection factor is shown in dark grey. Error bars were estimated using jackknife simulations. Figure 6. (a) Near-UV CD spectra of the F exch /U exch (continuous line), F s (broken line), U Gdn (dotted line), and calculated U exch (X) states. (b) Gdn denaturation of the F exch /U exch state monitored by near-uv CD; molar ellipticity at nm as a function of [Gdn]. NMR hydrogen-exchange experiments The strong evidence for burial of the Trp36 indole in the U exch state from this work as well as previous studies 16 led us to measure the accessibility of the Trp36 indole proton (N 11 H) to exchange with solvent. A 1.4 mm F exch /U exch equilibrium sample of the drkn SH3 domain was investigated at 5 8C and ph 7.6 using the CLEANEX approach (see Materials and Methods). Experimentally observed backbone amide proton exchange rates for the U exch state range from 0.3 s 21 (Ile48) to 12.0 s 21 (Asn51), while no significant exchange cross-peak could be observed for the Trp36 N 11 H. NH proton protection factors calculated on the basis of intrinsic exchange rate constants are shown in Figure Assuming an upper limit of 0.3 s 21 for the experimental exchange rate and an intrinsic exchange rate for a tryptophan N 11 H of 3.1 s 21, 21 a lower limit for the protection factor of about 10 was determined for the Trp36 N 11 H in the U exch state. The only other significantly protected NH positions are nearby in the primary sequence, Asn35 and Arg38, providing further evidence for burial of the Trp36 indole group in stable residual structure within the U exch state. NH hydrogen-exchange data for the F exch state of the drkn SH3 domain were obtained in the same experiment. Backbone amide NH protons were highly protected from solvent exchange (no exchange cross-peaks observed for 46 out of 57 backbone NH protons) as expected for a compact; hydrogen bonded and folded protein (data not shown). It is noteworthy that no exchange crosspeak was found for the Trp36 N 11 proton in the F exch state. Although the side-chain of Trp36 is located on the binding surface, the Trp36 N 11 proton may hydrogen bond to the Asp32 side-chain, leading to considerable protection from solvent exchange. Discussion NMR; total chemical shift change A large change in 15 N chemical shift reflects a significant change in the electronic and electrostatic structure surrounding the amide nitrogen atom associated with a specific residue. The greatest changes in 15 N chemical shift upon chemical or thermal denaturation of the U exch state are associated with residues in the region from 17 to 29 in the drkn SH3 domain (Figure 2). This is suggestive of residual (native or non-native) structure and ordered interactions in the U exch state that undergo substantial disruption with an increase in

8 170 Interactions and Buried Trp in Unfolded SH3 Domain temperature or concentration of Gdn. Previous evidence highlighted the presence of residual structure in this region of the molecule (summarised in Introduction). The chemical shift changes between U exch and denatured states of the drkn SH3 domain are highly significant and widespread across all residues in both experiments, more so than observed in denaturation experiments on other proteins. One example is a mutant form of protein L where only three of over 60 residues showed significant change in proton chemical shift between a compact denatured state and an extended conformation in 5 M guanidine. 7 Similarly, only six out of 80 amide protons showed any notable deviation from random coil chemical shift values for BPV-1 E2 DNA binding domain (which is unfolded in the absence of its DNA-binding partner). 22 Thus, when using chemical shift as a probe of structure, it is apparent that the U exch state of the drkn SH3 domain has significantly more residual structure than many other disordered proteins studied. The five isoleucine residues (I4, I24, I27, I48, and I53) have five of the top six largest chemical shift changes in the thermal denaturation. The x 1 torsion angle has a large influence on backbone 15 N shielding for valine, isoleucine, and leucine residues, 23,24 with the g-gauche rotamer giving rise to 15 N backbone resonance positions that are further upfield than other rotamers. Thermal denaturation of the drkn SH3 domain causes the isoleucine 15 N peaks to shift significantly upfield upon denaturation, suggesting that in the U exch state the isoleucine residues are participating in interactions that stabilise non-g-gauche rotomers. Upon denaturation, these interactions are eliminated, leading to averaged conformations of the isoleucine residues in the denatured state that increase the population of g-gauche rotomers, thereby resulting in the substantial upfield shift observed. The large change in chemical shift seen for these hydrophobic residues would be consistent with residual hydrophobic clustering in the U exch state that is interrupted upon denaturation. Changes in chemical shift may also reflect loss of hydrogen bonds when moving from the U exch to a denatured state. Wishart et al. have reported a strong correlation between hydrogen bond energies and amide proton or nitrogen chemical shifts. 25 This dependence is suggestive that any residues shown in Figure 2 to have a large chemical shift change are involved in residual backbone amide hydrogen bonding in the U exch state that is disrupted with increasing concentration of denaturant. In short, large changes in chemical shift between the unfolded and denatured states of the drkn SH3 domain suggest the presence of both hydrophobic and hydrogen bonding interactions within a stabilised core of the U exch state. It is interesting that the amide peaks for residues His7 and Asp8 weaken and disappear early in the temperature denaturation experiment. Broadening may be due to local conformational exchange between different substates in the U exch ensemble of structures, reflecting stabilised local structure. However, in 15 N relaxation dispersion experiments performed at 20 8C, no significant exchange contribution to the 15 N line-width was found, indicating that intermediate exchange would have to be faster than,10 4 s 21, depending on the populations of the various substates. Evidence for an interaction between the side-chains of His7 and Asp8 in the U exch state has been derived on the basis of pk a measurements (M.T. et al., manuscript in preparation). 26 Such a stabilising interaction might cause a notable increase in the local correlation time and hence contribute to the line width. Other evidence for intermediate exchange due to selective stabilisation of specific local conformations has been noted in the broadening of residues at 5 8C. 15 Both Gdn and thermal denaturation experiments reported here show that these residues display sigmoidal transitions when monitoring amide nitrogen chemical shift change as a function of denaturant, providing further evidence for stabilised local interactions in this region (see below). Evidence from NMR data for a specific region of residual structure in the U exch state Analysis of the chemical shift change as a function of temperature or Gdn concentration highlights a subset of residues for each data set that are best fit to a sigmoidal curve with a Hill coefficient $ 1.6 (Table 1, Figure 3(a) and (c)). In general, the denaturation of stable, fully folded proteins is thought to be a cooperative process (implying that the network of interactions stabilising the folded state structure is disrupted all at once) resulting in a steep sigmoidal transition between states. 27 We have interpreted the sigmoidal transitions observed upon denaturation of the U exch state to similarly represent cooperative interactions in the unfolded state that are disrupted in a concerted manner, causing local unfolding in this region. The shallower curve shapes for the denaturation of the U exch state suggest that the network of interactions is more transient or more localised compared to what is observed for a folded protein. Sigmoidal curves may not implicate interactions involving the sidechain of the particular residue directly; rather, they imply that the backbone atoms are probing interactions adjacent to them. These sigmoidal curves correspond to identical or nearby residues in the chemical and thermal denaturation. When residues with Hill coefficients $1.6 from the two denaturation experiments are indicated on a surface representation of the folded state of the drkn SH3 domain (Figure 8), most map to a contiguous area of the structure (even though some residues are far apart in sequence) corresponding to the b4 strand, the n-src loop into the b5 strand and residues in close contact with this region including some in the b6 strand and in the diverging turn in

9 Interactions and Buried Trp in Unfolded SH3 Domain 171 Figure 8. Surface of the folded state structure of the drkn SH3 domain. Residues involved in cooperative interactions in the unfolded state are coloured blue. The Figure was generated using Swiss-PdbViewer v3.7b2 56 and rendered using POV-Ray for Windows v3.1. the folded state. This suggests that the U exch state has residual native-like structure corresponding to the central b-sheet (b4 b5 b6). Interactions between residues in this sheet appear to melt cooperatively upon addition of denaturant, as illustrated by the sigmoidal-shaped curves (Figure 3). The clustering together within this sheet of residues with sigmoidal transitions further supports our interpretation of cooperativity. Similar interactions had been suggested by computational modelling methods indicating that the most highly populated structures in the U exch ensemble maintain the central b4 b6 strands. 28 Additionally, b4 b6 comprise one of the most stable regions during molecular dynamics unfolding simulations (M. Philippopoulos, J.D.F.-K. & R. Pomès, manuscript in preparation). This work provides an experimental demonstration of the presence of residual interactions in the central b-sheet. What does a sigmoidal transition in circular dichroism experiments imply? Sigmoidal transitions are observed during temperature denaturation monitored by far-uv CD for both the F exch /U exch and F s samples of the drkn SH3 domain (Figure 5). The F s state is more stable with t m almost 20 deg. C higher than the apparent melting temperature of the F exch /U exch sample. The cooperative, sigmoidal shape for the F exch / U exch melt is remarkable, however, since thermal melts are typically interpreted as a change from a 100% folded (upper baseline) to a 0% folded (lower baseline) state, and t m, by definition, is the point at which the protein is 50% folded and 50% unfolded in solution. Thus, it is dangerous to infer two-state behaviour from sigmoidal curves and, in particular, using only one experimental probe may lead to misinterpretation of the data. Since the ratio of F exch to U exch states at 5 8C is known from NMR studies to be approximately 55:45, a melting curve consisting of the second half of the sigmoidal curve would be expected. This implies the far-uv CD spectra of the F exch and U exch states likely resemble each other more closely than either resembles the thermally denatured U temp state (reached at the end of the experiment). The sigmoidal curve could reflect the cooperative loss of residual b-sheet structure in the U exch state superimposed on the loss of b-sheet structure in the folded state. The transition point, therefore, may represent the point at which 50% of cooperative b-structure remains in both folded and unfolded state ensembles. Evidence for greater structural similarity between F exch and U exch states compared to denatured states is also provided by near-uv CD spectra. A comparison of the calculated U exch spectrum to the U Gdn spectrum, representing a conformational state closer to random coil, shows that the U exch state has far more residual tertiary interactions involving the aromatics (Figure 6(a)). Peaks representing tyrosine and phenylalanine residues are clearly seen in the U exch state spectrum. The tryptophan peak (at 290 nm) is most striking, in that it has the same intensity as observed in the folded state, suggesting that the tryptophan is involved, on average, in an equal number of interactions in the unfolded and the folded states, with some unfolded substates having greater burial of the indole group than in the folded state and others less. A deconvolution of the thermodynamic parameters There are inherent difficulties in using thermodynamic equations (equations (2) (4) in Materials and Methods) to fit sigmoidal transitions from CD and fluorescence data for this complex system. These curve fits, and the resulting parameters, are based on the assumption that the denaturation transition is two-state, folded and unfolded. However, the U exch and U Gdn or U temp states are distinct. While disordered states of proteins differ structurally and energetically in the presence and in the absence of denaturants, 4 the drkn SH3 domain U exch state appears to be more distinct from denatured states than for many other proteins, raising the question of applicability of simplistic thermodynamic analysis. If the equations still apply, the calculated DG H2O represents an average of the free energy of denaturation of both F exch and U exch

10 172 Interactions and Buried Trp in Unfolded SH3 Domain states to U Gdn. Similarly, DH m represents an average of the change in enthalpy at t m for F exch and U exch denaturation. Table 2 lists the free energy values that have been defined here but note that these do not correspond to the DG H2O between the F exch and U exch states, which can be estimated at approximately 0.11 kcal mol 21 at 5 8C on the basis of NMR experimental data showing the ratio of F exch to U exch at this temperature (data not shown). If the DG H2O from near-uv CD data can be deconvoluted knowing that the ratio of folded to unfolded under non-denaturing conditions is,55:45, then an approximate DG H2O for the more structured components of the U exch state alone ( U exch ) relative to the less structured component ( U Gdn ) can be calculated in the absence of denaturant. The DG H2O between the F exch /U exch equilibrium state and U Gdn derived from the near-uv CD Gdn titration is 1.47 kcal mol 21 (Table 2), and since: DG H2O ¼ 2RT lnð½u Gdn Š=ð½F exch Šþð½ U exch Š þ½ U Gdn ŠÞÞÞ and: ð½f exch Šþð½ U exch Šþ½ U Gdn ŠÞÞ ¼ 0:55 þ 0:45 ¼ 1 then [ U Gdn ] ¼ 0.07 and U exch ¼ This implies that in the absence of denaturant: ½ U Gdn Š=½F exch Š¼0:07=0:55 and: ½ U Gdn Š=½U exch Š¼0:07=0:38: Therefore, DG H2O for F exch is 1.14 kcal mol 21, and for U exch is 0.93 kcal mol 21. These values further illustrate that the U exch state is much more similar (energetically, in this case) to the F exch state than to either U Gdn or U temp denatured states, with both the F exch and U exch states of the drkn SH3 domain being fairly unstable compared to other fully folded protein domains. Tryptophan and aromatic interactions The behaviour of the lone tryptophan residue (at position 36 in the drkn SH3 domain) in denaturation experiments is quite varied depending on the probe used. Fluorescence spectra indicate the F exch /U exch ensemble of states denatures noncooperatively, since the denaturation curve is not sigmoidal in shape (Figure 4). This contrasts to denaturation of the F s state, which yields a sigmoidal transition. Unfortunately, since the F exch /U exch experiment reflects the denaturation of a mixture of different protein conformations, it is very difficult to deconvolute the data obtained. The fluorescence results are perhaps the most difficult to interpret, since tryptophan fluorescence is influenced by many factors, including solvent. The fact that the fluorescence intensity increases upon denaturation by Gdn (as opposed to the more commonly observed solvent-mediated quenching) for both F s and F exch /U exch states suggests that factors other than local changes in polarity are contributing to the signal. For example, the close proximity of Tyr37 is likely to quench the Trp36 fluorescence when the protein is in a more compact conformation (either in the F exch or U exch state), while moving to a denatured state would relieve this quenching and cause an increase in fluorescence. These multiple competing influences might also have contributed in the Gdn denaturation experiment to a modification of the curve shape, thereby opening the possibility that the intrinsic change in indole environment is cooperative but that this behaviour has been obscured. NMR titration data for the Trp36 backbone amide proton shows no sigmoidal characteristics, while the side-chain indole amide 15 N resonance has only a marginally sigmoidal curve shape (see Table 1). This is somewhat surprising, given the current hydrogen-exchange and near-uv CD results and previous stop-flow fluorescence data 16 showing that the tryptophan residue is buried in the U exch state and therefore likely to be participating in hydrophobic interactions. Previous NMR cross-correlated relaxation experiments have provided evidence that the Trp36 side-chain is more rigid than other side-chains, with some immobilisation of other aromatic side-chains as well. 18 The lack of sigmoidal transitions for backbone NH groups of aromatic residues may be explained in that chemical shifts reflect only the local environment of the backbone atoms. If there is aromatic clustering in the unfolded state, it most likely involves the rings, so backbone NH atoms might not be a useful probe. The marginally sigmoidal transition for the indole side-chain of Trp36, however, is more difficult to understand. A denaturation experiment that follows the chemical shifts of side-chain carbon atoms in the aromatic rings may be more informative. Currently, experiments are being designed that will measure NOEs between aromatic ring protons in the U exch state. Hydrogen-exchange experiments provide further evidence for Trp indole burial in the U exch state The most intriguing result of the NH proton exchange experiments is the fact that no significant exchange cross-peaks were found for the sidechain indole proton of the single tryptophan residue (Trp36) in the U exch state of the drkn SH3 domain at ph 7.6 and 5 8C, leading to a lower limit for the protection factor of 10. A protection factor of 10 corresponds to a situation where a particular solvent-exchangeable proton is protected from exchange for 90% of the time, while it exchanges with its intrinsic exchange rate for 10% of the time. Therefore, the lower limit for the population of structures in which the tryptophan

11 Interactions and Buried Trp in Unfolded SH3 Domain 173 Figure 9. C a traces of the folded state structure (left) and representative structures from the four highest populated clusters in the U exch state (right, calculated by ENSEMBLE 28 ). The percentage population of each cluster is given adjacent to each representative structure. The residues corresponding to the seven b-strands of the folded state are coloured as follows: b1, red; b2, orange; b3, yellow; b4, green; b5, cyan; b6, dark blue; b7, magenta. The Trp36 sidechain is shown in order to illustrate the degree of burial in each representative structure. The Figure was generated using Swiss-PdbViewer v3.7b2 56 and rendered using POV-Ray for Windows v3.1. N 11 H is protected from exchange in the U exch state ensemble is,90%. In contrast, the experimental exchange rates (k obs ) for backbone amide protons generally correlate well with intrinsic exchange rates (k int ) predicted for fully exposed backbone amide protons. Hence, amide proton protection factors for the U exch state are clustered around a value of unity and range from 0.2 to 5.6 with an average of 1.12 ^ Considering an uncertainty factor of 2 3 for the prediction of the intrinsic amide proton exchange rates k int, 21,29 and the experimental uncertainty for k obs, which is indicated by the error bars in Figure 7, the majority of backbone amide protons in the unfolded state ensemble appear to be unprotected from exchange with solvent water. These results indicate that stable, hydrogen bonded elements of secondary structure are generally not present in the U exch ensemble of the drkn SH3 domain. While this conclusion is in agreement with 1 H a, 13 C a, 13 C b and 13 C 0 secondary chemical shifts, it may be considered surprising in light of the evidence for cooperative structure in the central b-sheet. It should be noted, however, that transient hydrogen bonding or transient burial of an amide proton results in rather small deviations of the protection factor from a value of 1 and can therefore hardly be identified due to the considerable uncertainties in the calculation of intrinsic exchange rate constants. The substantial protection of the Trp36 side-chain NH function from solvent exchange is, however, corroborated by the fact that the two most protected backbone amide protons (those of Arg38 and Asn35) are adjacent in the primary sequence of the protein. In comparison, no protection factors larger than 2.4 were observed for the denatured state of staphylococcal nuclease. 21 Applying the experimental results to structural models of the denatured ensemble In this study, we have gained more detailed information about residual structure in the U exch state of the drkn SH3 domain. There is now concrete NMR experimental evidence to support theoretical and modelling data that show residual structure in the central core of the

12 174 Interactions and Buried Trp in Unfolded SH3 Domain b-sheet. Near-UV CD and hydrogen-exchange experiments confirm that the single tryptophan residue in the domain is buried significantly and that there may be interactions among aromatic rings in the unfolded state. Chemical shift data indicate that residual hydrophobic clustering might involve aliphatic residues such as isoleucine and leucine. Upcoming experiments to monitor NOE distance restraints between aliphatic and aromatic side-chain groups should clarify the extent of hydrophobic clustering in the U exch state of the drkn SH3 domain. The specific structural data reported here will be a valuable addition to the experimental data pool used by ENSEMBLE, our recently developed program to generate ensembles of structures representing the unfolded state on the basis of experimental spectroscopic data. ENSEMBLE has already been applied to initial characterisation of the U exch state using primarily NH NH NOE and hydrodynamic data. 28 Input of cooperative interactions and protection factors involving tryptophan will provide a significant increase in the number of restraints applied to ENSEMBLE calculations. Utilising the large body of experimental data measured on the U exch state in concert with the ENSEMBLE approach is the most potent combination available to provide a clear picture of the structural features of this well-characterised disordered state, including specifics regarding hydrophobic clustering and the involvement of Trp36. Hydrophobic clustering within disordered states, in general, has been suggested to lead to collapse and to nucleate structure. 30,31 Characterisation of other unfolded or denatured states have highlighted hydrophobic clustering; 6,32,33 in particular, a recent report of clustering involving Trp62 in the unfolded state of lysozyme. 34 Evidence for non-native interactions in this case reinforce the concept that both native and non-native hydrophobic clustering occur in disordered states. Kinetic studies of the drkn SH3 domain suggest that non-native helical structure in the region from 16 to 28 can be stabilised by interactions with Trp36, 35 but an expanded native-like b-structurecontaining SH3 domain topology can accommodate a non-native burial of the Trp36 ring. Representative structures of groups of coordinates having significant population on the basis of ENSEMBLE calculations demonstrate that the Trp ring can be buried in a number of different structural contexts (see Figure 9). 28 This explains the hydrogen-exchange protection factor implicating 90% burial in the context of a highly heterogeneous and rapidly interconverting state. This extensive study of the U exch state of the drkn SH3 domain, which is highly populated under non-denaturing conditions, continues to lay a foundation for approaches to study other disordered states. Such characterisation is critical, as many proteins and domains are disordered under native conditions, particularly in the absence of binding targets. 1 Advances in the understanding of physiologically relevant disordered states can have important implications for the roles they play in molecular recognition in vivo, aswellasin a number of disease states. Materials and Methods Sample preparation The expression and purification of unlabelled, 15 N-labelled and N, C-labelled drkn SH3 domain were performed as described 16,36 with the following exceptions: (1) HMS 174(DE3) cells were used for expression; (2) the cells were lysed by stirring the resuspended solution with lysozyme (0.5 mg ml 21 ) for 20 minutes, and then with deoxycholic acid (1.2 mg ml 21 ) for five minutes, before a final sonication step. Yields of approximately 4 7 mg l 21 culture were obtained. High-purity aqueous 8 M guanidine hydrochloride was purchased from Pierce (Rockford, IL). NMR titrations NMR samples of the drkn SH3 domain in an equilibrium mixture of folded and unfolded states contained 1.3 mm 15 N-labelled and 0.5 mm 15 N, 3 C-labelled protein (for the chemical and thermal denaturation experiments, respectively) in 50 mm sodium phosphate (ph 6.0, 10% 2 H 2 O). These buffer conditions at 5 8C were used at the start of each set of titration experiments. NMR experiments were performed on a Varian UNITY Plus 500 MHz spectrometer equipped with triple-resonance pulsed field gradient probes with actively shielded z-gradients and gradient amplifier units. Data were processed and analysed on SGI stations and a Linux-based PC using NMRPipe/NMRDraw 37,38 and NMRView 39,40 software. Careful referencing of chemical shifts is critical. Due to its insensitivity to changes in temperature, DSS was used as a direct 1 H reference (at mm), as well as an indirect reference for 13 C and 15 N through a conversion involving J ratios. 41 Chemical denaturation Eleven 1 H 15 N HSQC spectra were recorded from 0 M to 2.7 M Gdn by addition of portions of 8 M stock solution. The experiment utilised 128 and 1024 complex points in t 1 and t 2, respectively, with a gradient sensitivity-enhanced approach. 42 Backbone resonance assignments for the drkn SH3 domain in 0 and 2 M Gdn at 5 8C have been reported. 15,43 The movement of peaks between these two assigned spectra could be followed directly, or inferred via their straight-line trajectory when obscured by overlap. In addition to the DSS reference, a correction was performed on the chemical denaturation data, since an increase in the concentration of salt due to the guanidinium and chloride ions can have a large effect on chemical shift, especially that of the amide nitrogen. Correction factors were obtained from Plaxco et al. 44 Although the published correction factors were determined for peptides studied at 20 8C and ph 5, they were applied directly to our system at 5 8C and ph 6.