The focus of structural biology is on studies of the highly populated, ground states of biological molecules; states that are only sparsely and transiently populated are more difficult to probe because they are invisible to most structural methods. Yet, such states can play critical roles in biochemical processes such as ligand binding, enzyme catalysis, and protein folding. A description of these states in terms of structure and dynamics is, therefore, of great importance. Here, we present a method, based on relaxation dispersion NMR spectroscopy of weakly aligned molecules in a magnetic field, that can provide such a description by direct measurement of backbone amide bond vector orientations in transient, low populated states that are not observable directly. Such information, obtained through the measurement of residual dipolar couplings, has until now been restricted to proteins that produce observable spectra. The methodology is applied and validated in a study of the binding of a target peptide to an SH3 domain from the yeast protein Abp1p and subsequently used in an application to protein folding of a mutational variant of the Fyn SH3 domain where 1 H-15 N dipolar couplings of the invisible unfolded state of the domain are obtained. The approach, which can be used to obtain orientational restraints at other sites in proteins as well, promises to significantly extend the available information necessary for providing a site-specific characterization of structural properties of transient, low populated states that have to this point remained recalcitrant to detailed analysis.olution NMR spectroscopy is a powerful technique for the study of biomolecular dynamics spanning a range of time scales from picoseconds for bond vector librations to many hours for hydrogen exchange in the buried interiors of proteins (1, 2). One very important approach, based on the concept of a ''spin-echo'' that was first described by Hahn in 1950 (3), is called the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method (4, 5). This class of experiment provides a window into processes with conformational exchange on the millisecond time scale (6), a time regime that is often the relevant one for the lifetimes of bound ligands (7,8), protein folding events (9), or molecular rearrangements that are important for the control of enzyme function (10-13). For systems in which the ground state exchanges with a minor conformer populated at 0.5% or higher and with exchange rates on the order of a hundred to a few thousand per second, the CPMG dispersion experiment provides a sensitive measure of the exchange dynamics (6). Rates of exchange, populations of exchanging states, and chemical shifts of nuclear spins in minor states can be obtained from fits of dispersion profiles to the appropriate model of exchange. Most importantly, information from potentially every residue is obtained in states that are often invisible in even the most sensitive of NMR spectra. Fig. 1a illustrates a simple case in which a loop of a protein, highlighted ...

SH3 domains, which are among the most frequently occurring protein interaction modules in nature, bind to peptide targets ranging in length from 7 to more than 25 residues. Although the bulk of studies on the peptide binding properties of SH3 domains have focused on interactions with relatively short peptides (less than 10 residues), a number of domains have been recently shown to require much longer sequences for optimal binding affinity. To gain greater insight into the binding mechanism and biological importance of interactions between an SH3 domain and extended peptide sequences, we have investigated interactions of the yeast Abp1p SH3 domain (AbpSH3) with several physiologically relevant 17-residue target peptide sequences. To obtain a molecular model for AbpSH3 interactions, we solved the structure of the AbpSH3 bound to a target peptide from the yeast actin patch kinase, Ark1p. Peptide target complexes from binding partners Scp1p and Sjl2p were also characterized, revealing that the AbpSH3 uses a common extended interface for interaction with these peptides, despite K d values for these peptides ranging from 0.3 to 6 M. Mutagenesis studies demonstrated that residues across the whole 17-residue binding site are important both for maximal in vitro binding affinity and for in vivo function. Sequence conservation analysis revealed that both the AbpSH3 and its extended target sequences are highly conserved across diverse fungal species as well as higher eukaryotes. Our data imply that the AbpSH3 must bind extended target sites to function efficiently inside the cell.Many protein interactions within signaling pathways are mediated by small modular domains, which are found within larger proteins (1). SH3 domains are one of the most frequently occurring of these protein-protein interaction modules in eukaryotic cells. These domains are ϳ60-residue ␤-sheet proteins that have been generally observed to bind to short proline-rich peptides containing the core consensus sequences ϩXXPXXP (class I) or PXXPXϩ (class II), where X can be a variety of residues, and ϩ is a Lys or Arg residue (2-4). SH3 domains often bind peptides with modest affinities (5-100 M (5)), and many SH3 domains appear to possess low specificity, binding to various PXXP-containing peptides with similar affinities (6 -10). These observations have led to the establishment of a "promiscuous model," which postulates that the signaling specificity of pathways depends primarily on factors other than the intrinsic binding properties of isolated SH3 domains (11), and that short peptide targets are likely sufficient for SH3 domain function. Arguing against this model, it has been shown that some SH3 domains require an extended target peptide (12-30 residues) to achieve maximal binding affinity (12-15). These results imply that the intrinsic specificity of SH3 domains may indeed play a significant role. The growing realization of the importance of interactions between SH3 domains and extended peptides provides the motivation for further studies to investigat...

There is increasing evidence for the functional importance of multiple dynamically populated states within single proteins. However, peptide binding by protein-protein interaction domains, such as the SH3 domain, has generally been considered to involve the full engagement of peptide to the binding surface with minimal dynamics and simple methods to determine dynamics at the binding surface for multiple related complexes have not been described. We have used NMR spectroscopy combined with isothermal titration calorimetry to comprehensively examine the extent of engagement to the yeast Abp1p SH3 domain for 24 different peptides. Over one quarter of the domain residues display co-linear chemical shift perturbation (CCSP) behavior, in which the position of a given chemical shift in a complex is co-linear with the same chemical shift in the other complexes, providing evidence that each complex exists as a unique dynamic rapidly inter-converting ensemble. The extent the specificity determining sub-surface of AbpSH3 is engaged as judged by CCSP analysis correlates with structural and thermodynamic measurements as well as with functional data, revealing the basis for significant structural and functional diversity amongst the related complexes. Thus, CCSP analysis can distinguish peptide complexes that may appear identical in terms of general structure and percent peptide occupancy but have significant local binding differences across the interface, affecting their ability to transmit conformational change across the domain and resulting in functional differences.

Structural biology is the study of the molecular arrangement and dynamics of biological macromolecules, particularly proteins. The resulting structures are then used to help explain how proteins function. This article gives the reader an insight into protein structure and the underlying chemistry and physics that is used to uncover protein structure. We start with the chemistry of amino acids and how they interact within, and between proteins, we also explore the four levels of protein structure and how proteins fold into discrete domains. We consider the thermodynamics of protein folding and why proteins misfold. We look at protein dynamics and how proteins can take on a range of conformations and states. In the second part of this review, we describe the variety of methods biochemists use to uncover the structure and properties of proteins that were described in the first part. Protein structural biology is a relatively new and exciting field that promises to provide atomic-level detail to more and more of the molecules that are fundamental to life processes.

We report the crystal structures and biophysical characterization of two stabilized mutants of the Drosophila Engrailed homeodomain that have been engineered to minimize electrostatic repulsion. Four independent copies of each mutant occupy the crystal lattice, and comparison of these structures illustrates variation that can be partly ascribed to networks of correlated conformational adjustments. Central to one network is leucine 26 (Leu 26 ), which occupies alternatively two side chain rotameric conformations (-gauche and trans) and different positions within the hydrophobic core. Similar sets of conformational substates are observed in other Engrailed structures and in another homeodomain. The pattern of structural adjustments can account for NMR relaxation data and sequence co-variation networks in the wider homeodomain family. It may also explain the dysfunction associated with a P26L mutation in the human ARX homeodomain protein. Finally, we observe a novel dipolar interaction between a conserved tryptophan and a water molecule positioned along the normal to the indole ring. This interaction may explain the distinctive fluorescent properties of the homeodomain family.The homeodomain is a simple fold common to many different DNA-binding proteins from diverse eukaryotes. The domain is found in transcription factors that regulate a variety of genes and so control key processes ranging from early developmental decision to homeostasis and general "housekeeping" (1-3).The homeodomain fold comprises three helices, connected by a flexible loop and a ␤-turn, and has a small hydrophobic core that is remarkably well conserved (4). The side chains in this conserved core appear to be well packed, which is a hallmark of stable folds. However, homeodomain folds generally have poorer stabilities in comparison with other proteins of similar size (5-8). The termini of homeodomains are disordered in solution (9 -11) and structures of free-and DNA-bound forms illustrate that DNA binding is accompanied by a structural condensation of the termini (12). Possibly, the homeodomain core also undergoes subtle structural changes upon DNA binding. Thus, the homeodomain would appear to mold to the surface of specific DNA by induced fit. Despite the wealth of current stereochemical and functional data for homeodomains, the basis for their comparatively low stability and its relationship, if any, to their induced fit remains to be established.Sequence co-variance analysis of homeodomains highlights several strongly correlated residue pairs that form conserved interactions and that may affect stability and DNA binding (4). One pair identified was the surface residues 17 and 52, which forms, in the majority of cases, a salt bridge. However, in the transcriptional repressor Engrailed from Drosophila, two lysines are instead found in these positions. We have substituted lysine 52 for glutamate, to mimic the conserved Glu 17 -Arg 52 salt bridge, and for alanine, to relieve the repulsion of the clustered positive charges. Standard equilibrium ...

Many protein-protein interaction domains bind to multiple targets. However, little is known about how the interactions of a single domain with many proteins are controlled and modulated under varying cellular conditions. In this study, we investigated the in vivo effects of Abp1p SH3 domain mutants that incrementally reduce target-binding affinity in four different yeast mutant backgrounds in which Abp1p activity is essential for growth. Although the severity of the phenotypic defects observed generally increased as binding affinity was reduced, some genetic backgrounds (prk1D and sla1D) tolerated large affinity reductions while others (sac6D and sla2D) were much more sensitive to these reductions. To elucidate the mechanisms behind these observations, we determined that Ark1p is the most important Abp1p SH3 domain interactor in prk1D cells, but that interactions with multiple targets, including Ark1p and Scp1p, are required in the sac6D background. We establish that the Abp1p SH3 domain makes different, functionally important interactions under different genetic conditions, and these changes in function are reflected by changes in the binding affinity requirement of the domain. These data provide the first evidence of biological relevance for any Abp1p SH3 domain-mediated interaction. We also find that considerable reductions in binding affinity are tolerated by the cell with little effect on growth rate, even when the actin cytoskeletal morphology is significantly perturbed.

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We report an unusual interaction in which a water molecule approaches the heterocyclic nitrogen of tryptophan and histidine along an axis that is roughly perpendicular to the aromatic plane of the side chain. The interaction is distinct from the well-known conventional aromatic hydrogen-bond, and it occurs at roughly the same frequency in protein structures. Calculations indicate that the water-indole interaction is favorable energetically, and we find several cases in which such contacts are conserved among structural orthologs. The indole-water interaction links side chains and peptide backbone in turn regions, connects the side chains in beta-sheets, and bridges secondary elements from different domains. We suggest that the water-indole interaction can be indirectly responsible for the quenching of tryptophan fluorescence that is observed in the folding of homeodomains and, possibly, many other proteins. We also observe a similar interaction between water and the imidazole nitrogens of the histidine side chain. Taken together, these observations suggest that the unconventional water-indole and water-imidazole interactions provide a small but favorable contribution to protein structures.