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

doi:10.1016/S0022-2836(02)00741-6 available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 322, 163–178

Cooperative Interactions and a Non-native Buried Trp in the Unfolded State of an SH3 Domain Karin A. Crowhurst1,2, Martin Tollinger1 and Julie D. Forman-Kay1,2* 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

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 (Uexch) state of the drkN SH3 domain is significantly populated and exists in equilibrium with the folded (Fexch) 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 Uexch 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

*Corresponding author

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; Uexch and Fexch, the unfolded and the folded state of the drkN SH3 domain; UGdn, the denatured state of the drkN SH3 domain in $2.0 M guanidinium chloride; Utemp, 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, 2,2dimethyl-2-sila-pentane-5-sulfonate; FID, free induction decay. E-mail address of the corresponding author: [email protected]

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

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

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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 (Fexch) and significantly populated unfolded (Uexch) 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

Interactions and Buried Trp in Unfolded SH3 Domain

nomenclature in this regard, with the unfolded (Uexch) state referring to the ensemble of structures of the drkN SH3 domain that exists in equilibrium with the folded (Fexch) 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 UGdn denatured state in $ 2.0 M Gdn and the Utemp denatured state at $ 70 8C. A large body of previous work has provided evidence for residual structure in the Uexch state of the drkN SH3 domain.13 – 17 The 1H – 15N 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 JHNHA coupling experiments indicate non-native a-helical propensity for residues 16 –20 in the Uexch state.15 The Uexch 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 Uexch state than the folded state, where it is located on the surface.16 Much of the previous characterisation of the Uexch state has focussed on comparison to the folded state of the drkN SH3 domain. Here, our primary approach is to compare the Uexch and denatured states. Differences between them imply that the Uexch 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 Uexch 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 Uexch 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

165

Interactions and Buried Trp in Unfolded SH3 Domain

sistent interactions and more extended structure that corresponds to the denatured state (UGdn or Utemp). In monitoring the chemical shift change for each residue from the Uexch 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 Fexch and Uexch contributions, and the data therefore represent an average of the two states. One approach to deconvoluting the two contributions is to compare the Fexch/Uexch denaturation data to experiments performed on the fully folded state (Fs) of the drkN SH3 domain, stabilised by the addition of 0.4 M Na2SO4. Since chemical shifts for residues in the Fexch and Fs 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 Fexch/Uexch states of the drkN SH3 domain, it is possible to infer the contribution of the Uexch state alone. NMR guanidine denaturation experiment

Figure 2. Maximal observed 15N chemical shift change for Uexch 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 N11 of Trp36.

transition to be monitored is between the Uexch and the denatured (UGdn or Utemp) states. As described previously,13 under non-denaturing conditions, the drkN SH3 domain can be found in equilibrium between a folded (Fexch) and a significantly populated unfolded (Uexch) 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 Fexch state and the other half to the Uexch state. It is due to the separation of the two states that NMR is such a powerful tool for studying the Uexch 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 Fexch peaks disappear and the Uexch peaks shift, reflecting a change in the populations of the conformations within the Uexch ensemble. The titration is complete when the peaks no longer shift. This new spectrum represents an ensemble of conformations with few per-

Gdn was titrated from 0 M to 2.7 M into a sample of 1.3 mM 15N-labelled drkN SH3 domain in the Fexch/Uexch states (50 mM sodium phosphate, pH 6). Upon addition of Gdn, Fexch peaks in the 1 H – 15N HSQC spectra disappear quickly (no Fexch peak is seen at 0.6 M), while the Uexch state peaks move. Their final positions represent the spectrum for the guanidine-denatured state of the drkN SH3 domain, UGdn. For HSQC spectra of both the Fexch/ Uexch equilibrium and the UGdn states, see Zhang et al.13 Amide nitrogen chemical shifts were used as reporters of structural changes during the transition between the Uexch and UGdn states. The overall 15N 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 15N 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

166

Interactions and Buried Trp in Unfolded SH3 Domain

Figure 3. (a) and (b) 15N chemical shift change from Uexch to UGdn states (normalised) as a function of [Gdn]. (c) and (d) 15N chemical shift change from Uexch to Utemp 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 to 70 8C 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 Fexch peaks in the 1 H – 15N HSQC spectra disappear (at about 50 8C, not as quickly as in the Gdn titration), and the

Uexch peaks again undergo a change in chemical shift. The unfolded resonances at the end of the thermal denaturation are representative of the Utemp state, and the chemical shifts do not correspond to those observed at the end of the Gdn experiment. This Utemp state is therefore distinct from both the Uexch and UGdn states. In plotting the overall 15N chemical shift change as a function of residue for this data (Figure 2(b)), the maximal chemical shift change was measured between the 15N 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 24 –28. 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

167

Interactions and Buried Trp in Unfolded SH3 Domain

Table 1. Summary of Hill coefficients derived from curve-fitting NMR titration data Residue E2 A3 I4 A5 K6 H7 D8 F9 S10 A11 T12 A13 D14 D15 E16 L17 S18 F19 R20 K21 T22 Q23 I24 L25 K26 I27 L28 N29 M30 E31 D32 D33 S34 N35 W36 W36 side-chain Y37 R38 A39 E40 L41 D42 G43 K44 E45 G46 L47 I48 S50 N51 Y52 I53 E54 M55 K56 N57 H58 D59

HGdna

R 2Gdnb

0.79 ^ 0.04 1.04 ^ 0.08 0.71 ^ 0.02 0.70 ^ 0.11 2.42 ^ 0.39c 0.73 ^ 0.07 0.51 ^ 0.34 0.70 ^ 0.03 1.06 ^ 0.18 0.76 ^ 0.06 1.05 ^ 0.15 0.81 ^ 0.05 1.24 ^ 0.09 1.21 ^ 0.10

1.000 0.999 1.000 0.997 0.997 0.999 0.967 1.000 0.996 0.999 0.997 0.999 0.999 0.998

1.54 ^ 0.20 1.03 ^ 0.08 1.09 ^ 0.10 1.28 ^ 0.10 1.74 ^ 0.18 1.81 ^ 0.33 1.97 ^ 0.21 2.91 ^ 0.26 2.76 ^ 0.73 1.99 ^ 0.83

0.995 0.999 0.998 0.998 0.997 0.992 0.997 0.998 0.976 0.948

1.28 ^ 0.32

0.980

0.85 ^ 0.13 0.71 ^ 0.04

0.998 1.000

0.86 ^ 0.50

0.986

0.79 ^ 0.20 0.44 ^ 0.11

0.990 0.996

2.00 ^ 0.57 0.56 ^ 0.20

0.973 0.989

0.74 ^ 0.21 0.62 ^ 0.16 1.07 ^ 0.45 0.63 ^ 0.06 0.56 ^ 0.06 0.90 ^ 0.06 0.82 ^ 0.12 0.91 ^ 0.14

0.993 0.994 0.978 0.999 0.999 0.999 0.996 0.998

Htempa

R 2tempb

1.15 ^ 0.13 1.13 ^ 0.11 1.00 ^ 0.07 2.08 ^ 0.28

1.000 1.000 1.000 0.996

1.11 ^ 0.24 0.85 ^ 0.27 1.12 ^ 0.12 1.08 ^ 0.12 0.97 ^ 0.10 1.25 ^ 0.26 1.31 ^ 0.19

0.999 0.999 1.000 0.999 1.000 0.998 0.998

1.13 ^ 0.28

0.997

1.25 ^ 0.27 1.61 ^ 0.31 0.92 ^ 0.26

0.998 0.994 0.999

0.98 ^ 0.11 1.00 ^ 0.25 1.50 ^ 0.10 2.18 ^ 0.10 1.35 ^ 0.20 1.20 ^ 0.08 1.62 ^ 0.08 1.83 ^ 0.15 1.31 ^ 0.13

1.000 0.998 1.000 1.000 0.998 1.000 1.000 0.999 0.999

3.17 ^ 1.05 1.23 ^ 0.16 1.08 ^ 0.11 2.57 ^ 0.28 1.10 ^ 0.14 1.35 ^ 0.22 1.36 ^ 0.09 1.06 ^ 0.15 1.18 ^ 0.16

0.969 0.999 1.000 0.997 0.999 0.998 1.000 0.999 0.999

1.37 ^ 0.47 1.15 ^ 0.19

0.994 0.999

1.24 ^ 0.15 1.43 ^ 0.18 3.93 ^ 1.03 1.16 ^ 0.07 1.03 ^ 0.16 0.88 ^ 0.75 1.36 ^ 0.14 1.14 ^ 0.08 1.20 ^ 0.07 0.86 ^ 0.14

0.999 0.999 0.972 1.000 0.999 0.992 0.999 1.000 1.000 1.000

0.98 ^ 0.58 0.98 ^ 0.58

0.996 0.996

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 Uexch state (with the exception of the extreme

Figure 4. Gdn denaturation experiments monitored by tryptophan fluorescence at 5 8C. Fexch/Uexch data (W) recorded at 358 nm, Fs 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 15N 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 tm values ranging from 31 8C to 42 8C 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 Fexch/Uexch equilibrium and Fs states (Figure 4). Under equilibrium conditions (Fexch/Uexch), chemical denaturation of the drkN SH3 domain does not produce a sigmoidal curve. Assuming a twostate denaturation, the Fs-derived sigmoidal curve was fit to a thermodynamic equation (equation (2) in Materials and Methods), from which the DGH2O (unfolding free energy in 0.4 M Na2SO4) 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 lmax 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.

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 Cm, Hilla (M Gdn)

HGdnb

R 2Hillc

DGH2Od (kcal mol21)

me

R 2thermc

1.08 ^ 0.10 3.31 ^ 0.18

0.995 0.996

– 2.18 ^ 0.06

– 1.39 ^ 0.04

– 0.999

Htempb

R 2Hill

DHmg (kcal mol21)

tm, thermf (8C)

R 2therm

5.20 ^ 0.20 9.72 ^ 0.27

0.995 0.997

16.1 ^ 1.6 33.1 ^ 1.1

35.7 ^ 1.3 60.1 ^ 0.3

0.996 0.998

C. Near-UV CD: Gdn denaturation monitoring Trp l HGdn Cm, Hill (M Gdn)

R 2Hill

DGH2O (kcal mol21)

m

R 2therm

Fexch/Uexch

0.996

1.47 ^ 0.14

3.48 ^ 0.27

0.995

Fexch/Uexch Fs

– 1.77 ^ 0.06

B. Far-UV CD: thermal denaturation tm, Hillf (8C) Fexch/Uexch Fs

a b c d e f g

38.4 ^ 0.3 59.6 ^ 0.2

0.46 ^ 0.01

2.87 ^ 0.19

Median transition concentration of Gdn. Hill coefficient for Gdn and thermal denaturation data, respectively. Coefficient of determination (quality of regression fit) for the Hill and thermodynamic equations, respectively. Free energy of unfolding or denaturation in water. The m-value: a measure of the protein’s sensitivity to denaturant. Median temperature of denaturation, calculated by Hill and thermodynamics equations, respectively. Enthalpy change at tm.

Equilibrium circular dichroism experiments Thermal denaturation of both the Fexch/Uexch and Fs states yield sigmoidal transitions reflecting loss of secondary structure when monitored by far-UV CD, although the tm (melting temperature) for the Fexch/Uexch states occurs at a lower temperature than that of the Fs 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 Fexch/Uexch and Fs data, since plots with non-horizontal baselines have overestimated Hill coefficient values. The tm and DHm values were derived (Table 2).

Figure 5. Thermal denaturation of the Fexch/Uexch state (W) and the Fs 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 Fexch/Uexch and Fs states to UGdn were not significant. Near-UV CD wavelength scans were performed on the Fexch/Uexch, Fs and UGdn 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 Fexch/Uexch 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 Fs and Fexch state structures are the same, subtraction of 55% of the Fs spectrum from the Fexch/Uexch spectrum should yield a spectrum representing the Uexch state. The three spectra for the Fs, Fexch/ Uexch and calculated Uexch 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 Fexch/Uexch 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 Fs state, possibly due to aggregation of the protein caused by high concentrations of protein and salt. Thermal denaturations monitored by nearUV CD for both states (Fexch/Uexch and Fs) were unsuccessful due to very small total changes in ellipticity.

169

Interactions and Buried Trp in Unfolded SH3 Domain

Figure 7. Protection factors from solvent exchange of backbone and Trp indole NH groups in the Uexch state as a function of residue number. Backbone NH protection factors are shown as black bars, while the lower limit for the tryptophan N11H protection factor is shown in dark grey. Error bars were estimated using jackknife simulations.

Figure 6. (a) Near-UV CD spectra of the Fexch/Uexch (continuous line), Fs (broken line), UGdn (dotted line), and calculated Uexch (X) states. (b) Gdn denaturation of the Fexch/Uexch state monitored by near-UV CD; molar ellipticity at 290.6 nm as a function of [Gdn].

NMR hydrogen-exchange experiments The strong evidence for burial of the Trp36 indole in the Uexch state from this work as well as previous studies16 led us to measure the accessibility of the Trp36 indole proton (N11H) to exchange with solvent. A 1.4 mM Fexch/Uexch 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 Uexch state range from 0.3 s21 (Ile48) to 12.0 s21 (Asn51), while no significant exchange cross-peak could be observed for the Trp36 N11H. NH proton protection factors calculated on the basis of intrinsic exchange rate constants are shown in Figure 7.20 Assuming an upper limit of 0.3 s21 for the experimental exchange rate and an intrinsic exchange rate for a tryptophan N11H of 3.1 s21,21 a lower limit for the protection factor of about 10 was determined for the Trp36 N11H in the Uexch 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 Uexch state. NH hydrogen-exchange data for the Fexch 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 N11 proton in the Fexch state. Although the side-chain of Trp36 is located on the binding surface, the Trp36 N11 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 15N 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 15N chemical shift upon chemical or thermal denaturation of the Uexch 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 Uexch state that undergo substantial disruption with an increase in

170

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 Uexch 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 Uexch 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 x1 torsion angle has a large influence on backbone 15N shielding for valine, isoleucine, and leucine residues,23,24 with the g-gauche rotamer giving rise to 15N backbone resonance positions that are further upfield than other rotamers. Thermal denaturation of the drkN SH3 domain causes the isoleucine 15N peaks to shift significantly upfield upon denaturation, suggesting that in the Uexch 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 Uexch state that is interrupted upon denaturation. Changes in chemical shift may also reflect loss of hydrogen bonds when moving from the Uexch 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 Uexch 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 Uexch 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

Interactions and Buried Trp in Unfolded SH3 Domain

between different substates in the Uexch ensemble of structures, reflecting stabilised local structure. However, in 15N relaxation dispersion experiments performed at 20 8C, no significant exchange contribution to the 15N line-width was found, indicating that intermediate exchange would have to be faster than , 104 s21, depending on the populations of the various substates. Evidence for an interaction between the side-chains of His7 and Asp8 in the Uexch state has been derived on the basis of pKa 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 23 – 28 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 Uexch 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 Uexch 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 Uexch 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

171

Interactions and Buried Trp in Unfolded SH3 Domain

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 v3.7b256 and rendered using POV-Ray for Windows v3.1.

the folded state. This suggests that the Uexch 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 Uexch 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. Pome`s, 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 Fexch/Uexch and Fs samples of the drkN SH3 domain (Figure 5). The Fs state is more stable with tm almost 20 deg. C higher than the apparent “melting temperature” of the Fexch/Uexch sample. The cooperative, sigmoidal shape for the Fexch/

Uexch 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 tm, 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 Fexch to Uexch 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 Fexch and Uexch states likely resemble each other more closely than either resembles the thermally denatured Utemp state (reached at the end of the experiment). The sigmoidal curve could reflect the cooperative loss of residual b-sheet structure in the Uexch 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 Fexch and Uexch states compared to denatured states is also provided by near-UV CD spectra. A comparison of the calculated Uexch spectrum to the UGdn spectrum, representing a conformational state closer to random coil, shows that the Uexch state has far more residual tertiary interactions involving the aromatics (Figure 6(a)). Peaks representing tyrosine and phenylalanine residues are clearly seen in the Uexch 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 Uexch and UGdn or Utemp 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 Uexch 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 DGH2O represents an average of the free energy of denaturation of both Fexch and Uexch

172

Interactions and Buried Trp in Unfolded SH3 Domain

states to UGdn. Similarly, DHm represents an average of the change in enthalpy at tm for Fexch and Uexch denaturation. Table 2 lists the free energy values that have been defined here but note that these do not correspond to the DGH2O between the Fexch and Uexch states, which can be estimated at approximately 0.11 kcal mol21 at 5 8C on the basis of NMR experimental data showing the ratio of Fexch to Uexch at this temperature (data not shown). If the DGH2O 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 DGH2O for the more structured components of the Uexch state alone (“Uexch”) relative to the less structured component (“UGdn”) can be calculated in the absence of denaturant. The DGH2O between the Fexch/Uexch equilibrium state and UGdn derived from the near-UV CD Gdn titration is 1.47 kcal mol21 (Table 2), and since: DGH2O ¼ 2RT lnð½UGdn =ð½Fexch  þ ð½“Uexch ” þ ½“UGdn ”ÞÞÞ and: ð½Fexch  þ ð½“Uexch ” þ ½“UGdn ”ÞÞ ¼ 0:55 þ 0:45 ¼ 1

then [“UGdn”] ¼ 0.07 and “Uexch” ¼ 0.38. implies that in the absence of denaturant:

This

½“UGdn ”=½Fexch  ¼ 0:07=0:55 and: ½“UGdn “=½Uexch  ¼ 0:07=0:38: Therefore, DGH2O for Fexch is 1.14 kcal mol21, and for Uexch is 0.93 kcal mol21. These values further illustrate that the Uexch state is much more similar (energetically, in this case) to the Fexch state than to either UGdn or Utemp denatured states, with both the Fexch and Uexch states of the drkN SH3 domain being fairly unstable compared to other fully folded protein domains.

monly observed solvent-mediated quenching) for both Fs and Fexch/Uexch 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 Fexch or Uexch 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 15N 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 data16 showing that the tryptophan residue is buried in the Uexch 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 Uexch state.

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 Fexch/Uexch ensemble of states denatures noncooperatively, since the denaturation curve is not sigmoidal in shape (Figure 4). This contrasts to denaturation of the Fs state, which yields a sigmoidal transition. Unfortunately, since the Fexch/Uexch 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 com-

Hydrogen-exchange experiments provide further evidence for Trp indole burial in the Uexch 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 Uexch 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

Interactions and Buried Trp in Unfolded SH3 Domain

173

Figure 9. Ca traces of the folded state structure (left) and representative structures from the four highest populated clusters in the Uexch state (right, calculated by ENSEMBLE28). 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 v3.7b256 and rendered using POV-Ray for Windows v3.1.

N11H is protected from exchange in the Uexch state ensemble is , 90%. In contrast, the experimental exchange rates (kobs) for backbone amide protons generally correlate well with intrinsic exchange rates (kint) predicted for fully exposed backbone amide protons. Hence, amide proton protection factors for the Uexch state are clustered around a value of unity and range from 0.2 to 5.6 with an average of 1.12 ^ 0.86. Considering an uncertainty factor of 2 – 3 for the prediction of the intrinsic amide proton exchange rates kint,21,29 and the experimental uncertainty for kobs, 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 Uexch ensemble of the drkN SH3 domain. While this conclusion is in agreement with 1Ha, 13Ca, 13Cb and 13C0 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 Uexch 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

174

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 Uexch 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 Uexch 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 Uexch 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 Uexch 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

Interactions and Buried Trp in Unfolded SH3 Domain

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, as well as in a number of disease states.

Materials and Methods Sample preparation The expression and purification of unlabelled, N-labelled and 15N, 13C-labelled drkN SH3 domain were performed as described16,36 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 ml21) for 20 minutes, and then with deoxycholic acid (1.2 mg ml21) for five minutes, before a final sonication step. Yields of approximately 4 – 7 mg l21 culture were obtained. High-purity aqueous 8 M guanidine hydrochloride was purchased from Pierce (Rockford, IL). 15

NMR titrations NMR samples of the drkN SH3 domain in an equilibrium mixture of folded and unfolded states contained 1.3 mM 15N-labelled and 0.5 mM 15N, 3C-labelled protein (for the chemical and thermal denaturation experiments, respectively) in 50 mM sodium phosphate (pH 6.0, 10% 2 H2O). 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/NMRDraw37,38 and NMRView39,40 software. Careful referencing of chemical shifts is critical. Due to its insensitivity to changes in temperature, DSS was used as a direct 1H reference (at 50 –200 mM), as well as an indirect reference for 13C and 15N through a conversion involving J ratios.41 Chemical denaturation Eleven 1H – 15N 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 t1 and t2, 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.

175

Interactions and Buried Trp in Unfolded SH3 Domain

Thermal denaturation A lower protein concentration (0.5 mM) was required in this experiment in order to avoid aggregation at high temperatures. 1H– 15N HSQC spectra, as described above, were recorded every 5 deg. C from 5 8C to 70 8C, with 128 and 512 complex points in t1 and t2, respectively. Additional experiments were required to assign spectra recorded between 5 8C and 30 8C, as well as above 30 8C. HNCO spectra45 recorded at 20, 30 and 50 8C utilised 54, 34 and 512 complex points in t1, t2 and t3, respectively, and were acquired with spectral widths of 1140.0, 1420.0 and 8000.0 Hz (F1, F2 and F3). The HNCO spectrum recorded at 40 8C utilised 64, 36 and 512 complex points in t1, t2 and t3, respectively, and was acquired with spectral widths of 1380.0, 1420.0 and 8000.0 Hz (F1, F2 and F3). Four scans were acquired for each free induction decay (FID) in all HNCO experiments. An HbCbCaCOCaHa experiment46 was recorded at 30 8C with eight scans acquired for each FID and spectral widths of 7649.6, 1140.0 and 6000.6 (F1, F2 and F3), with 54, 54 and 384 complex points in t1, t2 and t3, respectively. An HNCaCb experiment47 was recorded at 30 8C with 16 scans for each FID and spectral widths of 7649.6, 1420.0 and 8000.0 (F1, F2 and F3), with 54, 34 and 512 complex points in t1, t2 and t3, respectively. Once the HNCO was fully characterised at 30 8C, the movement of peaks could be tracked in order to assign the other HNCOs and subsequently the remaining HSQCs.

experiments were 100 mM drkN SH3 domain in 50 mM sodium phosphate buffer, pH 6 (Fexch/Uexch state), with a solution of 100 mM drkN SH3 domain in 6 M Gdn, 50 mM sodium phosphate (pH 6) used as the titrant. Protein ellipticity was monitored at 290.6 nm for the Fexch/Uexch state (the peak maximum for Trp in 0 M Gdn), with an equilibration time of two minutes before each recording and an averaging time of 30 seconds per datum point. Data analysis Hill equation Titration curves in all experiments were fit to the Hill equation in order to obtain a quantitative description of the degree of sigmoidal curvature: y ¼ y0 þ

axH þ xH

cH

ð1Þ

where y0 is the value of y at the start of the titration, a is a constant, x is either [Gdn] or temperature (in 8C), c is the value of x at the transition midpoint in the sigmoidal curve, and H is the Hill coefficient. When H ¼ 1, there is no cooperativity and the curve is hyperbolic. Increasing values of H above 1 indicate increasingly sigmoidal curves. Thermodynamics

Fluorescence spectroscopy; chemical denaturation Equilibrium denaturation of the drkN SH3 domain was monitored using a 1 cm path-length quartz cell at 5 8C with an AVIV Ratio spectrofluorometer model ATF105. Solutions of 5 mM drkN SH3 domain in 7.96 M Gdn, 50 mM sodium phosphate (pH 6) in the absence or in the presence of 0.4 M Na2SO4 were used as titrants. Sample excitation was at 295 nm, while emission was measured at maximum difference wavelengths of 358 nm and 369 nm, for the Fexch/Uexch (5 mM drkN SH3 domain in 50 mM sodium phosphate buffer, pH 6) and the Fs (5 mM drkN SH3 domain in 0.4 M Na2SO4, 50 mM sodium phosphate buffer, pH 6) samples, respectively. Far-UV circular dichroism; thermal denaturation Equilibrium denaturation of the drkN SH3 domain was monitored by far-UV CD spectroscopy with AVIV CD spectrometer model 62A DS, equipped with AVIV 60DS software v.4.1t. Thermal melts were performed on Fexch/Uexch (30 mM drkN SH3 domain in 50 mM sodium phosphate, pH 6) and Fs (30 mM drkN SH3 domain in 0.4 M Na2SO4, 50 mM sodium phosphate, pH 6) samples at 205 nm, between 5 8C and 89 8C, using a 0.1 cm pathlength quartz cell. Near-UV circular dichroism; chemical denaturation Near-UV CD spectroscopy was performed at 5 8C using a 1 cm path-length quartz cell with AVIV CD spectrometer model 62A DS, equipped with AVIV 60DS software v.4.1t. Full wavelength scans were performed on Fexch/Uexch (100 mM), Fs (100 mM) and UGdn (100 mM drkN SH3 domain in 50 mM sodium phosphate (pH 6), 3.5 M Gdn) between 250 and 300 nm, with an averaging time of 12 seconds in recording each datum point in 0.2 nm increments. Initial conditions for denaturation

If denaturation is approximated as two-state, sigmoidal curves can be fit to thermodynamic equations with regression analysis. For Gdn denaturation curves, an m-value and DGH2O (the free energy of unfolding or denaturation in the absence of denaturant) can be determined from:48 Iobs ¼ Ii 2 ðIi 2 If Þ

expððm½Gdn 2 DGH2O Þ=RTÞ ð2Þ 1 þ expððm½Gdn 2 DGH2O Þ=RTÞ

where Iobs is the observed intensity (fluorescence or ellipticity), Ii and If are the intensities of the initial and final states extrapolated to zero molar concentration of Gdn, respectively, R is the gas constant, 0.001987 kcal mol21 K21, and T is the temperature (278.15 K in all cases in these experiments). In thermal denaturation experiments, tm (the median temperature of denaturation) and DHm (the enthalpy change at tm) can be determined from fitting data to the following combined equations:49 Iobs ¼ ðIi 2 ðIi 2 If ÞÞðKt =ð1 þ Kt ÞÞ

ð3Þ



 2DHm ð1 2 t=tm Þ 2 DCp ððtm 2 tÞ þ t lnðt=tm ÞÞ Kt ¼ exp 2 RT ð4Þ where DCp ¼ 0.708 kcal mol21 K21 (12 cal mol21 K21 per amino acid residue).50 Hydrogen-exchange experiments NMR buffers contained 50 mM sodium phosphate (pH 7.6), 92% H2O/8% 2H2O, and the protein concentration of 15N-labelled drkN SH3 domain was 1.4 mM. All NMR spectra were recorded at 5 8C on a 800 MHz Varian Inova spectrometer. Amide proton exchange with solvent water for the Fexch and Uexch states was studied using a CLEANEX-PM pulse sequence, which

176

transfers magnetisation selectively from solvent water to protein NH groups for detection.51 Each 2D spectrum was recorded as a complex data matrix comprised of 96 £ 768 points. A total of 48 scans per FID were recorded, with a recycle delay of 1.5 s. A series of nine 2D spectra were collected with CLEANEX mixing times ranging from 2.5 ms to 68.3 ms. Data were processed and analysed using NMRPipe/NMRDraw software.38,52 Peak intensities were measured as the sum of the intensities for a 3 £ 3 grid centred on the peak maxima. A least-squares fitting procedure was employed to extract NH proton exchange rate constants, kobs, as described by Mori et al.21 and uncertainties were obtained from jackknife simulations.53 Numerical simulations were performed in order to assess the influence of exchange between Fexch and Uexch states on values of kobs. For this purpose, folding and unfolding rate constants were determined (as , 0.2 s21) employing a 15 N longitudinal relaxation experiment.54 Simulations using NH proton exchange rates within the experimentally determined range (between 0.3 s21 and 12.0 s21) show that contributions to kobs mediated by conformational exchange between Fexch and Uexch states during the CLEANEX mixing sequence are , 0.1 s21. NH proton protection factors were calculated as kint/kobs, where kint are the intrinsic exchange rate constants that were estimated using the method developed by Englander and co-workers.20

Acknowledgments The authors thank Dr Ranjith Muhandiram for NMR assistance, Drs Alan Davidson and Avi Chakrabartty for the use of their CD and fluorescence spectrometers, and Dr Yu-Keung Mok for technical assistance in protein expression and purification. In addition, we are grateful to Dr Wing-Yiu Choy for his aid with the comparative analysis of this data and his calculated ensemble structures, and to Drs David Wishart and Hue Sun Chan for helpful discussions with other analytical issues. K.A.C. is thankful to the Canadian Institutes of Health Research for a Doctoral Research Award, and M.T. is a recipient of an E. Schro¨dinger Fellowship (J-2086) of the Austrian Science Fund. This work was supported by funds from the Medical Research Council of Canada/Canadian Institutes of Health Research (to J.D.F.-K.).

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Interactions and Buried Trp in Unfolded SH3 Domain

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Edited by P. Wright (Received 27 March 2002; received in revised form 12 July 2002; accepted 15 July 2002)