Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states

The N-terminal SH3 domain of the Drosophila drk protein (drkN SH3) exists in equilibrium between folded and unfolded states under non-denaturing buffer conditions. In order to examine the origins of this instability, we have made mutations in the domain and characterized the thermodynamics and kinetics of folding. Results of substitutions of negatively charged residues to neutral amino acid residues suggest that the large electrostatic potential of the domain does not play a dominant role in the instability of the domain. Sequence alignment of a large number of SH3 domains reveals that the drkN SH3 domain has a threonine (T22) at a position corresponding to an otherwise highly conserved glycine residue in the diverging beta-turn connecting the beta3 and beta4 strands. Mutation of T22 to glycine results in significant stabilization of the drkN SH3 domain by 2.5 kcal/mole. To further characterize the basis for the stabilization of the T22 mutant relative to wild-type, we made additional mutant proteins with substitutions of residue T22. A strong correlation is seen between protein stability or folding rate and propensity for native beta-turn structure at this position. Correlation of folding rates with AGADIR predictions of non-native helical structure in the diverging turn region, along with our previous NMR evidence for non-native structure in this region of the unfolded state of the drkN SH3 domain, suggests that the free energy of the unfolded state also plays a role in stability. This result highlights the importance of both folded and unfolded states for understanding protein stability.     

Characterization of the hydrodynamic properties of the folding transition state of an SH3 domain by magnetization transfer NMR spectroscopy

Protein folding kinetic data have been obtained for the marginally stable N-terminal Src homology 3 domain of the Drosophila protein drk (drkN SH3) in an investigation of the hydrodynamic properties of its folding transition state. Due to the presence of NMR resonances of both folded and unfolded states at equilibrium, kinetic data can be derived from NMR magnetization transfer techniques under equilibrium conditions. Kinetic analysis as a function of urea (less than approximately 1 M) and glycerol enables determination of alpha values, measures of the energetic sensitivity of the transition state to the perturbation relative to the end states of the protein folding reaction (the folded and unfolded states). Both end states have previously been studied experimentally by NMR spectroscopic and other biophysical methods in great detail and under nondenaturing conditions. Combining these results with the kinetic folding data obtained here, we can characterize the folding transition state without requiring empirical models for the unfolded state structure. We are thus able to give a reliable measure of the solvent-accessible surface area of the transition state of the drkN SH3 domain (4730 +/- 360 A(2)) based on urea titration data. Glycerol titration data give similar results and additionally demonstrate that folding of this SH3 domain is dependent on solvent viscosity, which is indicative of at least partial hydration of the transition state. Because SH3 domains appear to fold by a common folding mechanism, the data presented here provide valuable insight into the transition states of the drkN and other SH3 domains.     

Cooperative interactions and a non-native buried Trp in the unfolded state of an SH3 domain

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 beta-sheet, particularly the beta4 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.     

NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions.

The N-terminal SH3 domain of drk (drkN SH3) is unstable, existing in equilibrium between a folded state (Fexch) and an unfolded state (Uexch) under non-denaturing buffer conditions. Using a15N/2H-labeled sample, long range amide NOEs can be observed in the Uexchstate as a result of reduced relaxation, in some cases correlating protons over 40 residues apart. These long range NOEs disappear upon addition of 2 M guanidinium chloride, demonstrating that there are substantial differences between the Uexchand the guanidine denatured states. Calculations using the long range NOEs of the Uexchstate yield highly compact structures having non-native turns and a non-native buried tryptophan residue. These structures agree with experimental stopped-flow fluorescence data and analytical ultracentrifugation results. Since protein stability depends on the structural and dynamic properties of both the folded and unfolded states, this study provides insights into the stability of the drkN SH3 domain. These results provide the first strong NOE-based evidence for compact unfolded states of proteins and suggest that some unfolded states under physiological conditions have specific interactions leading to compact structures.     

Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques

The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radius of gyration and hydrodynamic radius derived from the SAXS and PFG-NMR data, respectively, and (ii) a histogram of the size distribution of structures obtained from preliminary analyses of structural parameters recorded on the unfolded state. Results show that this unfolded state, U(exch), which exists in equilibrium with the folded state, F(exch), under non-denaturing conditions, is relatively compact, with the average size of conformers within the unfolded state ensemble only 30-40% larger than the folded state structure. In addition, the model predicts a significant overlap in the size range of structures comprising the U(exch) state with those in a denatured state obtained by addition of 2 M guanidinium chloride.     

Improved structural characterizations of the drkN SH3 domain unfolded state suggest a compact ensemble with native-like and non-native structure

Due to their dynamic ensemble nature and a deficiency of experimental restraints, disordered states of proteins are difficult to characterize structurally. Here, we have expanded upon our previous work on the unfolded state of the Drosophila drk N-terminal (drkN) SH3 domain with our program ENSEMBLE, which assigns population weights to pregenerated conformers in order to calculate ensembles of structures whose properties are collectively consistent with experimental measurements. The experimental restraint set has been enlarged with newly measured paramagnetic relaxation enhancements from Cu(2+) bound to an amino terminal Cu(2+)-Ni(2+) binding (ATCUN) motif as well as nuclear Overhauser effect (NOE) and hydrogen exchange data from recent studies. In addition, two new pseudo-energy minimization algorithms have been implemented that have dramatically improved the speed of ENSEMBLE population weight assignment. Finally, we have greatly improved our conformational sampling by utilizing a variety of techniques to generate both random structures and structures that are biased to contain elements of native-like or non-native structure. Although it is not possible to uniquely define a representative structural ensemble, we have been able to assess various properties of the drkN SH3 domain unfolded state by performing ENSEMBLE minimizations of different conformer pools. Specifically, we have found that the experimental restraint set enforces a compact structural distribution that is not consistent with an overall native-like topology but shows preference for local non-native structure in the regions corresponding to the diverging turn and the beta5 strand of the folded state and for local native-like structure in the region corresponding to the beta6 and beta7 strands. We suggest that this approach could be generally useful for the structural characterization of disordered states.     

Folding of a highly conserved diverging turn motif from the SH3 domain

Recent NMR structural characterization studies showed that a seven-residue segment (FKKGERL) from the src SH3 domain adopts the nativelike diverging type II beta-turn in aqueous solution in support of the prediction based on the I-sites library of sequence structural motifs. We study the conformational variability and folding/unfolding thermodynamics of this peptide in explicit solvent using replica-exchange molecular dynamics simulations, which greatly enhances the sampling of the conformational space. This peptide samples three main free energy basins (nativelike, intermediate, and unfolded) separated by small barriers. The nativelike basin is fractionally populated (DeltaG(300K) = 0.4 kcal/mol) with structures that satisfy a subset of the NMR-derived constraints. The intrinsic stability of the diverging turn is examined in relationship to the nature of three specific contacts: a turn-hydrogen bond, a mainchain-to-sidechain hydrogen bond, and an end-to-end hydrophobic contact. We have carried out simulations of mutants at the highly conserved GE positions in the sequence. The mutation E5D destabilizes the isolated diverging turn motif, contrary to the observation that this mutation stabilizes the fyn SH3 domain. The G4T mutation also destabilizes the isolated diverging turn; however, the extent of destabilization is smaller than that of the reverse mutation in the drk SH3.     

A 2D 13C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding

A 2D (13)C Chemical Exchange Saturation Transfer (CEST) experiment is presented for studying slowly exchanging protein systems using methyl groups as probes. The utility of the method is first established through studies of protein L, a small protein, for which chemical exchange on the millisecond time-scale is not observed. Subsequently the approach is applied to a folding exchange reaction of a G48M mutant Fyn SH3 domain, for which only cross-peaks derived from the folded ('ground') state are present in spectra. Fits of (15)N and methyl (13)C CEST profiles of the Fyn SH3 domain establish that the exchange reaction involves an interchange between folded and unfolded conformers, although elevated methyl (13)C transverse relaxation rates for some of the residues of the unfolded ('invisible, excited') state indicate that it likely exchanges with a third conformation as well. In addition to the kinetics of the exchange reaction, methyl carbon chemical shifts of the excited state are also obtained from analysis of the (13)C CEST data.     

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

Summary The backbone ¹H and ¹⁵N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the ¹⁵N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.Abbreviations 2D, 3D two-, three-dimensional - drkN SH3 N-terminal SH3 domain of Drosophila drk - HSQC heteronuclear single-quantum spectroscopy - NOE nuclear Overhauser enhancement - SH3 Src homology domain 3 - TOCSY total correlation spectroscopy     

Aromatic and methyl NOEs highlight hydrophobic clustering in the unfolded state of an SH3 domain

The N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) exists in equilibrium between a folded (F(exch)) state and a relatively compact unfolded (U(exch)) state under nondenaturing conditions. Selectively labeled samples of the domain have been analyzed by NOESY NMR experiments to probe residual hydrophobic clustering in the U(exch) state. The labeling strategy included selective protonation of aromatic rings or delta-methyl groups on Ile and Leu residues in a highly deuterated background. Combined with long mixing times, the methods permitted observation of significant numbers of long-range interactions between hydrophobic side chains, providing evidence for multiple conformers involving non-native hydrophobic clusters around the Trp 36 indole. Comparison of these data with previously reported HN-HN NOEs yields structural insight into the diversity of structures within the U(exch) ensemble in the drkN SH3 domain. Many of the HN-HN NOEs are consistent with models containing compact residual nativelike secondary structure and greater exposure of the Trp 36 indole to solvent, similar to kinetic intermediates formed in the hierarchic condensation model of folding. However, the methyl and aromatic NOE data better fit conformations with non-native burial of the Trp indole surrounded by hydrophobic groups and more loosely formed beta-structure; these structural characteristics are more consistent with those of kinetic intermediates formed during the hydrophobic collapse mechanism of folding. This suite of NOE data provides a more complete picture of the structures that span the U(exch) state ensemble, from conformers with non-native structure but long-range contacts to those that are highly nativelike. Together, the results are also consistent with the folding funnel view involving multiple folding pathways for this molecule.     

Analysis of a water mediated protein-protein interactions within RNase T1

Buried and well-ordered solvent molecules are an integral part of each folded protein. For a few individual water molecules, the exchange kinetics with solvent have been described in great detail. So far, little is known about the energetics of this exchange process. Here, we present an experimental approach to investigate water-mediated intramolecular protein-protein interactions by use of double mutant cycles. As a first example, we analyzed the interdependence of the contribution of two side chains (Asn9 and Thr93) to the conformational stability of RNase T1. In the folded state, both side chains are involved in the "solvation of the same water molecule WAT1. The coupling of the contributions of Asn9 and Thr93 to the conformational stability of RNase T1 was measured by urea unfolding and differential scanning calorimetry. The structural integrity of each mutant was analyzed by X-ray crystallography. We find that the effects of the Asn9Ala and the Thr93Ala mutations on the conformational stability are additive in the corresponding double mutant. We conclude that the free energy of the WAT1 mediated intramolecular protein-protein interaction in the folded state is very similar to solvent mediated protein-protein interaction in the unfolded state.     

Measuring the stability of partly folded proteins using TMAO

Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.     

Protein folding kinetics beyond the phi value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state

The SH3 domain folding transition state structure contains two well-ordered turn regions, known as the diverging turn and the distal loop. In the Src SH3 domain transition state, these regions are stabilized by a hydrogen bond between Glu30 in the diverging turn and Ser47 in the distal loop. We have examined the effects on folding kinetics of amino acid substitutions at the homologous positions (Glu24 and Ser41) in the Fyn SH3 domain. In contrast to most other folding kinetics studies which have focused primarily on non-disruptive substitutions with Ala or Gly, here we have examined the effects of substitutions with diverse amino acid residues. Using this approach, we demonstrate that the transition state structure is generally tolerant to amino acid substitutions. We also uncover a unique role for Ser at position 41 in facilitating folding of the distal loop, which can only be replicated by Asp at the same position. Both these residues appear to accelerate folding through the formation of short-range side-chain to backbone hydrogen bonds. The folding of the diverging turn region is shown to be driven primarily by local interactions. The diverging turn and distal loop regions are found to interact in the transition state structure, but only in the context of particular mutant backgrounds. This work demonstrates that studying the effects of a variety of amino acid substitutions on protein folding kinetics can provide unique insights into folding mechanisms which cannot be obtained by standard Phi value analysis.     

Effect of salt on the urea-unfolded form of barstar probed by m value measurements

To probe for residual structure present in the urea-unfolded form of the small protein barstar, to determine how salt might modulate such structure, and to determine how such structure might affect the stability of the protein, mutant variants that display m values different from that of the wild-type protein have been studied. The mutant proteins were obtained by site-directed mutagenesis at residue positions located on the surface of the folded protein. The m value, which represents the preferential free energy of interaction of urea with the unfolded form in comparison to that with the folded state, was determined from equilibrium urea-induced unfolding curves. Mutant proteins for which the m values were significantly greater than (m(+) mutant forms), significantly smaller than (m(-) mutant forms), or similar to (m(0) mutant forms) the m value determined for the wild-type protein were studied. The unfolded forms of the m(0), m(+) and m(-) mutant proteins represent different components within the unfolded form ensemble, which differ from each other in their solvent-exposed surface areas. Hence, the m value has been used as a measure of residual structure in the unfolded form. To further understand the nature of structures present in the unfolded form ensemble, the effects of the salt KCl on the stabilities of the wild-type and the mutant proteins, as well as on the structures present in the unfolded form ensemble, were also studied. It was found that the m values of the m(0), m(+) and m(-) mutant proteins all converge to the wild-type m value in the presence of KCl. This result indicates that the salt modulates residual structure in the unfolded form by screening electrostatic interactions that maintain compact and expanded components in the unfolded protein ensemble. The use of free energy cycles has allowed the effect of salt on the structure and free energy of the unfolded protein to be related to the stability of the protein.     

Effect of protein structural integrity on cross-linking by tyrosinase evidenced by multidimensional heteronuclear magnetic resonance spectroscopy

Enzymatic cross-linking of proteins can be catalyzed either by transferase-type enzymes, e.g., transglutaminases, or by oxidoreductases, e.g., tyrosinases or laccases. Three-dimensional structure of protein substrate plays a key role in these reactions, that is, the reactivity and end product are strongly modulated by the accessibility of target amino acid residues to the cross-linking enzyme. Typically structural integrity of protein can be distorted by heat, pH, or mechanical action, as well as by varying ionic concentration of the solution. In this study we used partially unfolded protein (wild-type DrkN SH3) and its structurally stabilized mutant (T22G) to investigate the impact of folded/unfolded conformations on cross-linking by Trichoderma reesei tyrosinase. Our results clearly showed formation of intermolecular cross-links solely between unfolded conformations, making them superior substrates to folded proteins when using tyrosinase as a cross-linking enzyme. Multidimensional heteronuclear magnetic resonance experiments in solution state were employed to investigate cross-linked end-products. The results presented in this study form basis for application development in food, medical, cosmetic, textile, packing and other sectors. In addition, the outcome of this study has a high value for the basic understanding of reaction mechanism of tyrosinases on proteins.     

Folding subdomains of thioredoxin characterized by native-state hydrogen exchange

Native-state hydrogen exchange (HX) studies, used in conjunction with NMR spectroscopy, have been carried out on Escherichia coli thioredoxin (Trx) for characterizing two folding subdomains of the protein. The backbone amide protons of only the slowest-exchanging 24 amino acid residues, of a total of 108 amino acid residues, could be followed at pH 7. The free energy of the opening event that results in an amide hydrogen exchanging with solvent (DeltaG(op)) was determined at each of the 24 amide hydrogen sites. The values of DeltaG(op) for the amide hydrogens belonging to residues in the helices alpha(1), alpha(2), and alpha(4) are consistent with them exchanging with the solvent only when the fully unfolded state is sampled transiently under native conditions. The denaturant-dependences of the values of DeltaG(op) provide very little evidence that the protein samples partially unfolded forms, lower in energy than the unfolded state. The amide hydrogens belonging to the residues in the beta strands, which form the core of the protein, appear to have higher values of DeltaG(op) than amide hydrogens belonging to residues in the helices, suggesting that they might be more stable to exchange. This apparently higher stability to HX of the beta strands might be either because they exchange out their amide hydrogens in a high energy intermediate preceding the globally unfolded state, or, more likely, because they form residual structure in the globally unfolded state. In either case, the central beta strands-beta(3,) beta(2), and beta(4)-would appear to form a cooperatively folding subunit of the protein. The native-state HX methodology has made it possible to characterize the free energy landscape that Trx can sample under equilibrium native conditions.     

Protein folding transition states probed by loop extension

We propose a new way to characterize protein folding transition states by (1) insertion of one or more residues into an unstructured protein loop, (2) measurement of the effect on protein folding kinetics and thermodynamics, and (3) analysis of the results in terms of a rate-equilibrium free energy relationship, alpha(Loop). alpha(Loop) reports on the fraction of molecules that form the perturbed loop in the transition state. Interpretation of the changes in equilibrium free energy using standard polymer theory can help detect residual structure in the unfolded state. We illustrate our approach with data for the model proteins CI2 and the alpha spectrin SH3 domain.     

Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches

The Thr29 residue in the hydrophobic core of goat alpha-lactalbumin (alpha-LA) was substituted with Val (Thr29Val) and Ile (Thr29Ile) to investigate the contribution of Thr29 to the thermodynamic stability of the protein. We carried out protein stability measurements, X-ray crystallographic analyses, and free energy calculations based on molecular dynamics simulation. The equilibrium unfolding transitions induced by guanidine hydrochloride demonstrated that the Thr29Val and Thr29Ile mutants were, respectively, 1.9 and 3.2 kcal/mol more stable than the wild-type protein (WT). The overall structures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the side-chain O-H group of Thr29 and the main-chain C=O group of Glu25. To analyze the stabilization mechanism of the mutants, we performed free energy calculations. The calculated free energy differences were in good agreement with the experimental values. The stabilization of the mutants was mainly caused by solvation loss in the denatured state. Furthermore, the O-H group of Thr29 favorably interacts with the C=O group of Glu25 to form hydrogen bonds and, simultaneously, unfavorably interacts electrostatically with the main-chain C=O group of Thr29. The difference in the free energy profile of the unfolding path between WT and the Thr29Ile mutant is discussed in light of our experimental and theoretical results.     

15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide ¹⁵N is used as a probe of solvent exchange. The experiment is performed in 50% H₂O–50% D₂O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, ¹⁵N^D, into protonated species, ¹⁵N^H, as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of ¹⁵N^H/D-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5–2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.