The native state conformational ensemble of the SH3 domain from alpha-spectrin.

The folding/unfolding equilibrium of the alpha-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the beta-strands, the short 3(10) helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the beta-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53-57) and a highly unstable region, the loop between residues 34-41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential scanning calorimetry, indicating that under our experimental conditions the amide exchange of all residues in the SH3 domain occurs primarily via local unfolding reactions. A structure-based thermodynamic analysis has allowed us to predict correctly the thermodynamics of the global unfolding of the domain and to define the ensemble of conformational states that quantitatively accounts for the observed pattern of hydrogen exchange protection. These results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium. The observation that specific regions of a protein are able to undergo independent local folding/unfolding reactions indicates that under native conditions the scale of cooperative interactions is regional rather than global.     

Cooperative propagation of local stability changes from low-stability and high-stability regions in a SH3 domain

Site-directed mutagenesis has been used to produce local stability changes at two regions of the binding site surface of the alpha-spectrin SH3 domain (Spc-SH3) differing in their intrinsic stability. Mutations were made at residue 56, located at the solvent-exposed side of the short 3(10) helix, and at residue 21 in the tip of the flexible RT-loop. NMR chemical-shift analysis and X-ray crystallography indicated negligible changes produced by the mutations in the native structure limited to subtle rearrangements near the mutated residue and at flexible loops. Additionally, mutations do not alter importantly the SH3 binding site structure, although produce significant changes in its affinity for a proline-rich decapeptide. The changes in global stability measured by differential scanning calorimetry are consistent the local energy changes predicted by theoretical models, with the most significant effects observed for the Ala-Gly mutations. Propagation of the local stability changes throughout the domain structure has been studied at a per-residue level of resolution by NMR-detected amide hydrogen-deuterium exchange (HX). Stability propagation is remarkably efficient in this small domain, apparently due to its intrinsically low stability. Nevertheless, the HX-core of the domain is not fully cooperative, indicating the existence of co-operative subunits within the core, which is markedly polarized. An equilibrium phi-analysis of the changes in the apparent Gibbs energies of HX per residue produced by the mutations has allowed us to characterize structurally the conformational states leading to HX. Some of these states resemble notably the folding transition state of the Spc-SH3 domain, suggesting a great potential of this approach to explore the folding energy landscape of proteins. An energy perturbation propagates more effectively from a flexible region to the core than in the opposite direction, because the former affects a broader region of the energy landscape than the latter. This might be of importance in understanding the special thermodynamic signature of the SH3-peptide interaction and the relevance of the dual character of SH3 binding sites.     

Dynamics of the Tec‐family tyrosine kinase SH3 domains

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains.     

Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core

Recent studies have shown that trans-phosphorylation of the Abl SH3 domain at Tyr89 by Src-family kinases is required for the full transforming activity of Bcr-Abl. Tyr89 localizes to a binding surface of the SH3 domain that engages the SH2-kinase linker in the crystal structure of the c-Abl core. Displacement of SH3 from the linker is likely to influence efficient downregulation of c-Abl. Hydrogen-deuterium exchange (HX) and mass spectrometry (MS) were used to investigate whether Tyr89 phosphorylation affects the ability of the SH3 domain to interact intramolecularly with the SH2-kinase linker in cis as well as other peptide ligands in trans. HX MS analysis of SH3 binding showed that when various Abl constructs were phosphorylated at Tyr89 by the Src-family kinase Hck, SH3 was unable to engage a high-affinity ligand in trans and that interaction with the linker in cis was reduced dramatically in a construct containing the SH3 and SH2 domains plus the linker. Phosphorylation of the Abl SH3 domain on Tyr89 also interfered with binding to the negative regulatory protein Abi-1 in trans. Site-directed mutagenesis of Tyr89 and Tyr245, another tyrosine phosphorylation site located in the linker that may also influence SH3 binding, implicated Tyr89 as the key residue necessary for disrupting regulation after phosphorylation. These results imply that phosphorylation at Tyr89 by Src-family kinases prevents engagement of the Abl SH3 domain with its intramolecular binding partner leading to enhanced Abl kinase activity and cellular signaling.     

The temperature dependence of the hydrogen exchange in the SH3 domain of alpha-spectrin

The amide hydrogen-deuterium exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin has been measured by nuclear magnetic resonance as a function of temperature between 8 and 46 degrees C. The analysis of the temperature dependence of HX from a statistical thermodynamic point of view has allowed us to estimate the enthalpies and entropies of the conformational processes leading to HX. The results indicate that under native conditions the domain undergoes a wide variety of conformational fluctuations, ranging from local motions, mainly located in loops, turns and chain ends and involving only low enthalpy and entropy, to extensive structural disruptions affecting its core and involving enthalpies and entropies that come fairly close to those observed during global unfolding.     

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.     

Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding

The HIV-1 accessory protein Nef controls multiple aspects of the viral life cycle and host immune response, making it an attractive therapeutic target. Previous X-ray crystal structures of Nef in complex with key host cell binding partners have shed light on protein–protein interactions critical to Nef function. Crystal structures of Nef in complex with either the SH3 or tandem SH3–SH2 domains of Src-family kinases reveal distinct dimer conformations of Nef. However, the existence of these Nef dimer complexes in solution has not been established. Here we used hydrogen exchange mass spectrometry (HX MS) to compare the solution conformation of Nef alone and in complexes with the SH3 or the SH3–SH2 domains of the Src-family kinase Hck. HX MS revealed that interaction with the Hck SH3 or tandem SH3–SH2 domains induces protection of the Nef αB-helix from deuterium uptake, consistent with a role for αB in dimer formation. HX MS analysis of a Nef mutant (position Asp123, a site buried in the Nef:SH3 dimer but surface exposed in the Nef:SH3–SH2 complex), showed a Hck-induced conformational change in Nef relative to wild-type Nef. These results support a model in which Src-family kinase binding induces conformational changes in Nef to expose residues critical for interaction with the μ1 subunit of adaptor protein 1 and the major histocompatibility complex-1 tail, and subsequent major histocompatibility complex-1 downregulation and immune escape of HIV-infected cells required for functional interactions with downstream binding partners.     

Structure of a helically extended SH3 domain of the T cell adapter protein ADAP

The adapter protein ADAP (FYB/SLAP-130) provides a critical link between T cell receptor (TCR) signaling and cell adhesion via the activation of integrins. The C-terminal 70 residues of ADAP show homology to SH3 domains; however, conserved residues of the fold are absent. An alignment and annotation of this domain has therefore been elusive. We have solved the three-dimensional structure of the ADAP C-terminal domain by NMR spectroscopy and show that it represents an altered SH3 domain fold. An N-terminal, amphipathic helix makes extensive contacts to residues of the regular SH3 domain fold, and thereby a composite surface with unusual surface properties is created. We propose this SH3 domain variant to be classified as a helically extended SH3 domain (hSH3 domain) and show that the ADAP-hSH3 domain can no longer bind conventional proline-rich peptides.     

The 1.1 A resolution crystal structure of the p130cas SH3 domain and ramifications for ligand selectivity

The Crk-associated tyrosine kinase substrate p130cas (CAS) is a docking protein containing an SH3 domain near its N terminus, followed by a short proline-rich segment, a large central substrate domain composed of 15 repeats of the four amino acid sequence YxxP, a serine-rich region and a carboxy-terminal domain, which possesses consensus binding sites for the SH2 and SH3 domains of Src (YDYV and RPLPSPP, respectively). The SH3 domain of CAS mediates its interaction with several proteins involved in signaling pathways such as focal adhesion kinase (FAK), tyrosine phosphatases PTP1B and PTP-PEST, and the guanine nucleotide exchange factor C3G. As a homolog of the corresponding Src docking domain, the CAS SH3 domain binds to proline-rich sequences (PxxP) of its interacting partners that can adopt a polyproline type II helix. We have determined a high-resolution X-ray structure of the recombinant human CAS SH3 domain. The domain, residues 1-69, crystallized in two related space groups, P2(1) and C222(1), that provided diffraction data to 1.1 A and 2.1 A, respectively. The crystal structure shows, in addition to the conserved SH3 domain architecture, the way in which the CAS characteristic amino acids form an atypically charged ligand-binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by the CAS SH3 domain. The structure enables modelling of the docking interactions to its ligands, for example from focal adhesion kinase, and supports structure-based drug design of inhibitors of the CAS-FAK interaction.     

Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin.

The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues. It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions. The function is unknown, but it is probably involved in specific protein--protein interactions. Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution. The crystals have two SH3 molecules per asymmetric unit. These two Fyn SH3 domains are not related by a local twofold axis. The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold. A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA. The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn). These loops are different in tyrosine kinase and spectrin SH3 domains. They could modulate the binding properties of the aromatic surface.     

Probing the nature of interactions in SH2 binding interfaces--evidence from electrospray ionization mass spectrometry.

We have adopted nanoflow electrospray ionization mass spectrometry (ESI-MS) and isothermal titration calorimetry (ITC) to probe the mechanism of peptide recognition by the SH2 domain from the Src family tyrosine kinase protein, Fyn. This domain is involved in the mediation of intracellular signal transduction pathways by interaction with proteins containing phosphorylated tyrosine (Y*) residues. The binding of tyrosyl phosphopeptides can mimic these interactions. Specificity in these interactions has been attributed to the interaction of the Y* and residues proximal and C-terminal to it. Previous studies have established that for specific binding with Fyn, the recognition sequence consists of pTyr-Glu-Glu-Ile. The specific interactions involve the binding of Y* with the ionic, and the Y* + 3 Ile residue with the hydrophobic binding pockets on the surface of the Fyn SH2 domain. In this work, a variation in the Y* + 3 residue of this high-affinity sequence was observed to result in changes in the relative binding affinities as determined in solution (ITC) and in the gas phase (nanoflow ESI-MS). X-ray analysis shows that a feature of the Src family SH2 domains is the involvement of water molecules in the peptide binding site. Under the nanoflow ESI conditions, water molecules appear to be maintained in the Fyn SH2-ligand complex. Compelling evidence for these molecules being incorporated in the SH2-peptide interface is provided by the prevalence of the peaks assigned to water-bound over the water-free complex at high-energy conditions. Thus, the stability of water protein-ligand complex appears to be intimately linked to the presence of water.     

Investigation of the four cooperative unfolding units existing in the MICAL-1 CH domain

The solution structure of human MICAL-1 calpolnin homology (CH) domain is composed of six alpha helices and one 3(10) helix. To study the unfolding of this domain, we carry out native-state hydrogen exchange, intrinsic fluorescence and far-UV circular dichroism experiments. The free energy of unfolding, DeltaG(H2O), is calculated to be 7.11+/-0.58 kcal mol(-1) from GuHCl denaturation at pH 6.5. Four cooperative unfolding units are found using native-state hydrogen exchange experiment. Forty-seven slow-exchange residues can be studied by native-state hydrogen exchange experiments. From the concentration dependence of exchange rates, free energy of amide hydrogen with solvent, DeltaG(HX) and m-value (sensitivity of exposure to denaturant) are obtained, which reveal four cooperative unfolding units. The slowest exchanging protons are distributed throughout the whole hydrophobic core of the protein, which might be the folding core. These results will help us understand the structure of MICAL-1 CH domain more deeply.     

Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip

The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.     

Measuring rapid hydrogen exchange in the homodimeric 36 kDa HIV-1 integrase catalytic core domain

Measurements of rapid hydrogen exchange (HX) of water with protein amide sites contain valuable information on protein structure and function, but current NMR methods for measuring HX rates are limited in their applicability to large protein systems. An alternate method for measuring rapid HX is presented that is well-suited for larger proteins, and we apply the method to the deuterated, homodimeric 36 kDa HIV-1 integrase catalytic core domain (CCD). Using long mixing times for water-amide magnetization exchange at multiple pH values, HX rates spanning more than four orders of magnitude were measured, as well as NOE cross-relaxation rates to nearby exchangeable protons. HX protection factors for the CCD are found to be large (>10(4)) for residues along the dimer interface, but much smaller in many other regions. Notably, the catalytic helix (residues 152-167) exhibits low HX protection at both ends, indicative of fraying at both termini as opposed to just the N-terminal end, as originally thought. Residues in the LEDGF/p75 binding pocket also show marginal stability, with protection factors in the 10-100 range (～1.4-2.7 kcal/mol). Additionally, elevated NOE cross-relaxation rates are identified and, as expected, correspond to proximity of the amide proton to a rapidly exchanging proton, typically from an OH side chain. Indirect NOE transfer between H(2) O and the amide proton of I141, a residue in the partially disordered active site of the enzyme, suggests its proximity to the side chain of S147, an interaction seen in the DNA-bound form for a homologous integrase.     

Stability and folding of the SH3 domain of bruton's tyrosine kinase

Bruton's tyrosine kinase (BTK) plays an important role in B cell development. Deletion of C-terminal 14 amino acids of the SH3 domain of BTK results in X-linked agammaglobulinemia (XLA), an inherited disease. We report here on the stability and folding of SH3 domain of BTK. Peptides corresponding to residues 216–273 (58 residues) and 216–259 (44 residues) of BTK SH3 domain were synthesized by solid phase methods; the first peptide constitutes the entire SH3 domain of BTK while the latter peptide lacks 14 amino acid residues of the C-terminal. The 58 amino acid peptide forms mainly a β-barrel type folding unit. Although small and lacking disulfide bonds, this peptide is extremely stable to thermal denaturation. Based on circular dichroism measurements, its melting temperature was found to be high, 82°C at pH 6.0. However, the Gibbs free energy (ΔG) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 2.6 kcal/mol by Gdn·HCl denaturation experiments, as compared to 12 kcal/mol obtained for larger single domain proteins, indicating poor stability of SH3 domain. Addition of 500 mM of Na₂SO₄ increased the free energy change ΔG to 4.0 kcal/mol, suggesting an ionic strength effect. The truncated peptide fails to fold correctly and adopts random coil conformation in contrast to 58 amino acid β-barrel peptide, which exhibits high thermal stability but normal or low stability at ambient temperature. These results, to our knowledge the first to delineate the importance of C-terminal in structural integrity of SH3 domains, indicate also that improper folding and/or poor stability of mutant SH3 domain in BTK likely causes XLA. Proteins 28:465–471 © 1996 Wiley-Liss, Inc.     

Hydrogen exchange kinetics of RNase A and the urea:TMAO paradigm

A key paradigm in the biology of adaptation holds that urea affects protein function by increasing the fluctuations of the native state, while trimethylamine N-oxide (TMAO) affects function in the opposite direction by decreasing the normal fluctuations of the native ensemble. Using urea and TMAO separately and together, hydrogen exchange (HX) studies on RNase A at pH* 6.35 were used to investigate the basic tenets of the urea:TMAO paradigm. TMAO (1 M) alone decreases HX rate constants of a select number of sites exchanging from the native ensemble, and low urea alone increases the rate constants of some of the same sites. Addition of TMAO to urea solutions containing RNase A also suppresses HX rate constants. The data show that urea and TMAO independently or in combination affect the dynamics of the native ensemble in opposing ways. The results provide evidence in support of the counteraction aspect of the urea:TMAO paradigm linking structural dynamics with protein function in urea-rich organs and organisms. RNase A is so resistant to urea denaturation at pH* 6.35 that even in the presence of 4.8 M urea, the native ensemble accounts for >99.5% of the protein. An essential test, devised to determine the HX mechanism of exchangeable protons, shows that over the 0-4.8 M urea concentration range nearly 80% of all observed sites convert from EX2 to EX1. The slow exchange sites are all EX1; they do not exhibit global exchange even at urea concentrations (5.8 M) well into the denaturation transition zone, and their energetically distinct activated complexes leading to exchange gives evidence of residual structure. Under these experimental conditions, the use of DeltaG(HX) as a basis for HX analysis of RNase A urea denaturation is invalid.     

Crystal structure of the N-terminal SH3 domain of mouse betaPIX, p21-activated kinase-interacting exchange factor

The mouse betaPIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3(2)21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact beta-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse betaPIX-SH3 domain binding the way in which the betaPIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse betaPIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse betaPIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of betaPIX-SH3 domain resulting in its multiple biological functions.     

Abl N-terminal cap stabilization of SH3 domain dynamics

Crystal structures and other biochemical data indicate that the N-terminal cap (NCap) region of the Abelson tyrosine kinase (c-Abl) is important for maintaining the downregulated conformation of the kinase domain. The exact contributions that the NCap makes in stabilizing the various intramolecular interactions within c-Abl are less clear. While the NCap appears to be important for locking the SH3 and SH2 domains to the back of the kinase domain, there may be other more subtle elements of regulation. Hydrogen exchange (HX) and mass spectrometry (MS) were used to determine if the NCap contributes to intramolecular interactions involving the Abl SH3 domain. Under physiological conditions, the Abl SH3 domain underwent partial unfolding and its unfolding half-life was slowed during binding to the SH2 kinase linker, providing a unique assay for testing NCap-induced stabilization of the SH3 domain in various constructs. The results showed that the NCap stabilizes the dynamics of the SH3 domain in certain constructs but does not increase the relative affinity of the SH3 domain for the native SH2 kinase linker. The stabilization effect was absent in constructs of just the NCap and SH3 but was obvious when the SH2 domain and the SH2 kinase linker were present. These results suggest that interactions between the NCap and the SH3 domain can contribute to c-Abl stabilization in constructs that contain at least the SH2 domain, an effect that may partially compensate for the absence of the negative regulatory C-terminal tail found in the related Src family of kinases.     

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.

We have determined the thermodynamic stability and peptide binding affinity of the carboxy-terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal-transduction protein Sem-5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation--at pH 7.3, the protein has a Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 degrees C. We have also characterized binding of the domain to 2 different length proline-rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem-5's likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2-fold increase in fluorescence intensity. Both bind with only modest affinities (Kd approximately equal to 30 microM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly-L-proline in a similar fashion.