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Vincent J Hilser

Publications and source records attributed to Vincent J Hilser.

16 recordsLinked to original sources
PubMed2006

Revealing the nature of the native state ensemble through cold denaturation.

Recent advances in NMR methodology have enabled the structural analysis of proteins at temperatures far below the freezing point of water, thus opening a window to the cold denaturation process. Although the phenomenon of cold denaturation has been known since the mid-1970s, the freezing point of water has prevented detailed and structurally resolved studies without application of additional significant perturbations of the protein ensemble. As a result, the cold-denatured state and the process of cold denaturation have gone largely unstudied. Here, the structural and thermodynamic basis of cold denaturation is explored with emphasis placed on the insights that are uniquely ascertained from low-temperature studies. It is shown that the noncooperative cold-induced unfolding of protein results in the population of partially folded states that cannot be accessed by other techniques. The structurally resolved view of the cold denaturation process therefore can provide direct access to the cooperative substructures within the protein molecule and provide an unprecedented structurally resolved picture of the states that comprise the native state ensemble.

Animals↗
PubMed2006

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding.

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.

Acids↗
PubMed2006

Ensemble-based signatures of energy propagation in proteins: a new view of an old phenomenon.

The ability of a protein to transmit the energetic effects of binding from one site to another constitutes the underlying basis for allosterism and signal transduction. Despite clear experimental evidence indicating the ability of proteins to transmit the effects of binding, the means by which this propagation is facilitated is not well understood. Using our previously developed ensemble-based description of the equilibrium, we investigated the physical basis of energy propagation and identified several fundamental and general aspects of energetic coupling between residues in a protein. First, partitioning of a conformational ensemble into four distinct sub-ensembles allows for explanation of the range of experimentally observed coupling behaviors (i.e., positive, neutral, and negative coupling between various regions of the protein structure). Second, the relative thermodynamic properties of these four sub-ensembles define the energetic coupling between residues as either positive, neutral, or negative. Third, analysis of the structural and thermodynamic features of the states within each sub-ensemble reveals significant variability. This third result suggests that a quantitative description of energy propagation in proteins requires an understanding of the structural and energetic features of more than just one or a few low-energy states, but also of many high-energy states. Such findings illuminate the difficulty in interpreting energy propagation in proteins in terms of a structural pathway that physically links coupled sites.

Binding Sites↗
PubMed2005

COREX/BEST server: a web browser-based program that calculates regional stability variations within protein structures.

SUMMARY: Utilizing the user-supplied coordinates of a protein structure, the COREX/BEST Server generates a structural thermodynamic ensemble. This conformational ensemble can then be used to calculate the regional variations in stability of a protein structure, and the stabilities are presented in units of energy (kcal/mol). The regional stabilities, which are calculated at the resolution of individual residues, can be mapped onto the protein structure for visual representation and downloaded from the site in the form of tab delimited text. The site provides an easy to follow summary of the theoretical and algorithmic approaches and provides links to references for more detailed descriptions. AVAILABILITY: The COREX/BEST Server may be accessed through a typical web browser by visiting http://best.utmb.edu/BEST/.

Algorithms↗
PubMed2005

Local conformational fluctuations can modulate the coupling between proton binding and global structural transitions in proteins.

Local conformational fluctuations in proteins can affect the coupling between ligand binding and global structural transitions. This finding was established by monitoring quantitatively how the population distribution in the ensemble of microstates of staphylococcal nuclease was affected by proton binding. Analysis of acid unfolding and proton-binding data with an ensemble-based model suggests that local fluctuations: (i) can be effective modulators of ligand-binding affinities, (ii) are important determinants of the cooperativity of ligand-driven global structural transitions, and (iii) are well represented thermodynamically as local unfolding processes. These studies illustrate how an ensemble-based description of proteins can be used to describe quantitatively the interdependence of local conformational fluctuations, ligand-binding processes, and global structural transitions. This level of understanding of the relationship between conformation, energy, and dynamics is required for a detailed mechanistic understanding of allostery, cooperativity, and other complex functional and regulatory properties of macromolecules.

Binding Sites↗
PubMed2004

An experimental strategy to evaluate the thermodynamic stability of highly dynamic binding sites in proteins using hydrogen exchange.

Quantitative characterization of the stability of highly dynamic regions in proteins is a significant goal because it represents a cornerstone to an understanding of the role of dynamics in function. Due to experimental constraints, however, monitoring the local stability of highly dynamic regions using standard hydrogen exchange (HX) methods is not a viable approach. Here, an experimental strategy is outlined that takes advantage of the coupling between stability as monitored by HX and binding affinity as monitored by isothermal titration calorimetry. It is shown that the stability of dynamic regions, which are part of binding sites, can be inferred from the response of the system to Gly mutations at surface-exposed sites. When applied to the analysis of the highly dynamic RT loop of SEM5 C-terminal SH3 domain, this approach reveals that the energetic consequences of the observed conformational heterogeneity are significant.

Binding Sites↗
PubMed2004

Thermodynamic mechanism and consequences of the polyproline II (PII) structural bias in the denatured states of proteins.

A quantitative characterization of the structure and energy of the denatured states of proteins represents the cornerstone to a molecular-level understanding of both protein stability and fold specificity. Recent studies have revealed a significant bias in unstructured peptides toward the polyproline II (P(II)) conformation, even when no prolines are present in the sequence. This indicates that the P(II) conformation is a dominant component of the denatured states of proteins, although a quantitative description of the component enthalpy and entropy functions associated with this conformation (i.e., the thermodynamic mechanism) has thus far proven elusive. An experimental system has been designed that, when analyzed with high-precision isothermal titration calorimetry, provides direct access to the residue-specific thermodynamics of the P(II) structure formation in disordered proteins and peptides. Here, it is shown that the P(II) bias is driven by a favorable and significant enthalpy (Deltah) of -1.7 kcal mol(-1) residue(-1), which is partially offset by an unfavorable entropy (TDeltas) of -0.7 kcal mol(-1) residue(-1), relative to the ensemble of disordered conformations of the molecule. In addition to impacting dramatically the interpretation of thermal denaturation experiments, these experimental values form the framework of a quantitative energetic description of the denatured states of proteins.

Animals↗
PubMed2004

Thermodynamics of binding to SH3 domains: the energetic impact of polyproline II (PII) helix formation.

Although numerous biophysical studies have focused on elucidating the structural and thermodynamic determinants that govern the free energy of binding between various SH3 domains and their putative recognition sequences, a quantitative accounting of the energetics of this interaction has proven enigmatic. Specifically, the binding results in a large and negative change on the standard enthalpy and entropy functions, a result which is inconsistent with the positive values for these quantities that is expected from the hydrophobic nature of the binding pocket. Here, the binding of the C-terminal SH3 domain of Sem-5 to its putative recognition peptide on the Sos (Son of Sevenless) protein is investigated using isothermal titration calorimetry under a variety of temperature and pH conditions. In addition, the energy associated with folding the Sos peptide into the binding competent polyproline II conformation is quantitatively evaluated. These results provide a rationale for the observed discrepancy between the experimental and predicted behavior and indicate that the determinants of binding in this system cannot be ascertained from a static structural representation of the binding process.

Amino Acid Sequence↗
PubMed2004

Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation.

The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching -35 degrees C. The cold-induced unfolding of ubiquitin is found to be highly noncooperative, in distinct contrast to the thermal melting of this and other proteins. These results demonstrate the potential of cold denaturation as a means to dissect the cooperative substructures of proteins and to provide a rigorous framework for testing statistical thermodynamic treatments of protein stability, dynamics and function.

Algorithms↗
PubMed2004

Directed discovery of bivalent peptide ligands to an SH3 domain.

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Analysis of the "thermodynamic information content" of a Homo sapiens structural database reveals hierarchical thermodynamic organization.

Classification of the amounts and types of lower order structural elements in proteins is a prerequisite to effective comparisons between protein folds. In an effort to provide an additional vehicle for fold comparison, we present an alternative classification scheme whereby protein folds are represented in statistical thermodynamic terms in such a way as to illuminate the energetic building blocks within protein structures. The thermodynamic relationship is examined between amino acid sequences and the conformational ensembles for a database of 159 Homo sapiens protein structures ranging from 50 to 250 amino acids. Using hierarchical clustering, it is shown through fold-recognition experiments that (1) eight thermodynamic environmental descriptors sufficiently accounts for the energetic variation within the native state ensembles of the H. sapiens structural database, (2) an amino acid library of only six residue types is sufficient to encode >90% of the thermodynamic information required for fold specificity in the entire database, and (3) structural resolution of the statistically derived environments reveals sequential cooperative segments throughout the protein, which are independent of secondary structure. As the first level of thermodynamic organization in proteins, these segments represent the thermodynamic counterpart to secondary structure.

Computer Simulation↗
PubMed2003

Solution structure, dynamics, and thermodynamics of the native state ensemble of the Sem-5 C-terminal SH3 domain.

Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Here the native state ensemble of the C-terminal src homology domain-3 (C-SH3) from Caenorhabditis elegans Sem-5 has been studied using a variety of high-resolution biophysical techniques. In addition to determining the first solution structure of the unliganded protein, we have performed (15)N relaxation and native state hydrogen-deuterium exchange. It is observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted by the COREX algorithm to be unfolded in many of the most probable conformational states within the native state ensemble. The implications of this agreement and the potential role of conformational heterogeneity of the observed biophysical properties are discussed.

Algorithms↗
PubMed2003

The effect of the polyproline II (PPII) conformation on the denatured state entropy.

Polyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM-5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to the conformational entropy change of the substituted amino acids. Results indicate that the denatured ensemble can be characterized by at least two thermodynamically distinct states, the PPII conformation and an unfolded state conforming to the previously held idea of the denatured state as a random collection of conformations determined largely by hard-sphere collision. The probability of the PPII conformation in the denatured states for Ala and Gly were found to be significant, approximately 30% and approximately 10%, respectively, resulting in a dramatic reduction in the conformational entropy of folding.

Alanine↗
PubMed2003

Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling.

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.

Binding Sites↗
PubMed2002

Thermodynamic environments in proteins: fundamental determinants of fold specificity.

To investigate the relationship between an amino acid sequence and its corresponding protein fold, a database of thermodynamic stability information was assembled as a function of residue type from 81 nonhomologous proteins. This information was obtained using the COREX algorithm, which computes an ensemble-based description of the native state of proteins. Dissection of the COREX stability constant into its fundamental energetic components resulted in 12 thermodynamic environments describing the tertiary architecture of protein folds. Because of the observation that residue types partitioned unequally between these environments, it was hypothesized that thermodynamic environments contained energetic information that connected sequence to fold. To test the significance of this hypothesis, the thermodynamic stability information was incorporated into a three-dimensional-to-one-dimensional scoring matrix, and simple fold recognition experiments were performed in a manner such that information about the fold target was never included in the scoring. For 60 out of 81 fold targets, the correct sequence for the target scored in the top 5% of 3858 decoy sequences, with Z-scores ranging from 1.76 to 12.23. Furthermore, a scoring matrix assembled from the residues of 40 nonhomologous all-alpha proteins was used to thread sequences against 12 nonhomologous all-beta protein targets. In 10 of 12 cases, sequences known to adopt the native all-beta structure scored in the top 5% of 3858 decoy sequences, with Z-scores ranging from 1.99 to 7.94. These results indicate that energetic information encoded by thermodynamic environments represents a fundamental property of proteins that underlies classifications based on secondary structure.

Algorithms↗