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David Shortle

Publications and source records attributed to David Shortle.

16 recordsLinked to original sources
PubMed2007

Characterization of denatured proteins using residual dipolar couplings.

The conventional view of denatured proteins as random coils is being challenged by nuclear magnetic resonance (NMR) experiments that can measure the relative orientation of backbone segments with respect to each other in a distance-independent manner. Known as the residual dipolar coupling (RDC), this NMR parameter is measurable on proteins that have been weakly aligned inside a NMR tube. The simple observation of dipolar couplings unequivocally establishes that a denatured protein is not a random coil. Structures of denatured states under differing experimental conditions can be compared by a simple scatter plot of the RDCs measured under each condition; direct structural interpretation, however, is much more problematic. Here some of the technical issues for achieving weak alignment of denatured proteins are addressed using three types of media: strained polyacrylamide gels, both neutral or highly charged, and neutral lipid liquid crystalline bicelles. All three alignment media are experimentally robust and compatible with 8 M urea and low pH, conditions frequently used to denature proteins.

Acrylic Resins↗
PubMed2006

Conformational preference of polyglycine in solution to elongated structure.

Do polypeptide chains ever behave like a random coil? In this report we demonstrate that glycine, the residue with the fewest backbone restrictions, exhibits a strong preference for an extended conformation in solution when polymerized in short segments of polyglycine. A model peptide system comprised of two unique tripeptide units, between which 1 to 18 glycine residues are inserted, is characterized by NMR and by small-angle X-ray scattering (SAXS). The residual dipolar coupling (RDC) values of the two tripeptide units are insensitive to changes in number of intervening glycines, suggesting that extension of the linker does not alter the average angular relationship between the tripeptides. Polyglycine segments longer than nine residues form insoluble aggregates. SAXS measurements using synchrotron radiation provide direct evidence that polyglycine peptides adopt elongated conformations. In particular, the construct with a linker with six glycines showed a scattering profile indicative of a monomeric state with a radius of gyration and the maximum dimension of 9.1 A and approximately 34 A, respectively. The ensemble averaged global structure of this 12-mer peptide can best be approximated by a cylinder with a radius of 4 A and a length of approximately 33 A, making it intermediate in extension between a beta strand and an alpha helix.

Amino Acid Sequence↗
PubMed2006

Multiple alignment tensors from a denatured protein.

The structural content of the denatured state has yet to be fully characterized. In recent years, large residual dipolar couplings (RDCs) from denatured proteins have been observed under alignment conditions produced by bicelles and strained polyacrylamide gels. In this report, we describe efforts to extend our picture of the residual structure in denatured nuclease by measuring RDCs with multiple alignment tensors. Backbone amide 15N-1H RDCs were collected from 4 M urea for a total of eight RDC data sets. The RDCs were analyzed by singular value decomposition (SVD) to determine the number of independent alignment tensors present in the data. On the basis of the resultant singular values and propagated error estimates, it is clear that there are at least three independent alignment tensors. These three independent RDC datasets can be reconstituted as orthogonal linear combinations, (OLC)-RDC datasets, of the eight actually recorded. The first, second, and third OLC-RDC datasets are highly robust to the removal of any single experimental RDC dataset, establishing the presence of three independent alignment tensors, sampled well above the level of experimental uncertainty. The observation that the RDC data span three or more dimensions of the five-dimensional parameter space demonstrates that the ensemble average structure of denatured nuclease must be asymmetric with respect to these three orthogonal principal axes, which is not inconsistent with earlier work demonstrating that it has a nativelike topology.

Bacteriophage Pf1↗
PubMed2006

Protein refolding in silico with atom-based statistical potentials and conformational search using a simple genetic algorithm.

A distance-dependent atom-pair potential that treats long range and local interactions separately has been developed and optimized to distinguish native protein structures from sets of incorrect or decoy structures. Atoms are divided into 30 types based on chemical properties and relative position in the amino acid side-chains. Several parameters affecting the calculation and evaluation of this statistical potential, such as the reference state, the bin width, cutoff distances between pairs, and the number of residues separating the atom pairs, are adjusted to achieve the best discrimination. The native structure has the lowest energy for 39 of the 40 sets of original ROSETTA decoys (1000 structures per set) and 23 of the 25 improved decoys (approximately 1900 structures per set). Combined with the orientation-dependent backbone hydrogen bonding potential used by ROSETTA and a statistical solvation potential based on the solvent exclusion model of Lazaridis & Karplus, this potential is used as a scoring function for conformational search based on a genetic algorithm method. After unfolding the native structure by changing every phi and psi angle by either +/-3, +/-5 or +/-7 degrees, five small proteins can be efficiently refolded, in some cases to within 0.5 A C(alpha) distance matrix error (DME) to the native state. Although no significant correlation is found between the total energy and structural similarity to the native state, a surprisingly strong correlation exists between the radius of gyration and the DME for low energy structures.

Algorithms↗
PubMed2005

A consistent set of statistical potentials for quantifying local side-chain and backbone interactions.

The frequencies of occurrence of atom arrangements in high-resolution protein structures provide some of the most accurate quantitative measures of interaction energies in proteins. In this report we extend our development of a consistent set of statistical potentials for quantifying local interactions between side-chains and the polypeptide backbone, as well as nearby side-chains. Starting with phi/psi/chi1 propensities that select for optimal interactions of the 20 amino acid side-chains with the 2 flanking peptide bonds, the following 3 new terms are added: (1) a distance-dependent interaction between the side-chain at i and the carbonyl oxygens and amide protons of the peptide units at i +/- 2, i +/- 3, and i +/- 4; (2) a distance-dependent interaction between the side-chain at position i and side-chains at positions i + 1 through i + 4; and (3) an orientation-dependent interaction between the side-chain at position i and side-chains at i + 1 through i + 4. The relative strengths of these 4 pseudo free energy terms are estimated by the average information content of each scoring matrix and by assessing their performance in a simple fragment threading test. They vary from -0.4 - -0.5 kcal/mole per residue for phi/psi/chi1 propensities to a range of -0.15 - -0.6 kcal/mole per residue for each of the other 3 terms. The combined energy function, containing no interactions between atoms more than 4 residues apart, identifies the correct structural fragment for randomly selected 15 mers over 40% of the time, after searching through 232,000 alternative conformations. For 14 out of 20 sets of all-atom Rosetta decoys analyzed, the native structure has a combined score lower than any of the 1700-1900 decoy conformations. The ability of this energy function to detect energetically important details of local structure is demonstrated by its power to distinguish high-resolution crystal structures from NMR solution structures.

Amino Acids↗
PubMed2005

Enhanced sampling near the native conformation using statistical potentials for local side-chain and backbone interactions.

In the preceding article in this issue of Proteins, an empirical energy function consisting of 4 statistical potentials that quantify local side-chain-backbone and side-chain-side-chain interactions has been demonstrated to successfully identify the native conformations of short sequence fragments and the native structure within large sets of high-quality decoys. Because this energy function consists entirely of interactions between residues separated by fewer than 5 positions, it can be used at the earliest stage of ab initio structure prediction to enhance the efficiency of conformational search. In this article, protein fragments are generated de novo by recombining very short segments of protein structures (2, 4, or 6 residues), either selected at random or optimized with respect this local energy function. When local energy is optimized in selected fragments, more efficient sampling of conformational space near the native conformation is consistently observed for 450 randomly selected single turn fragments, with turn lengths varying from 3 to 12 residues and all 4 combinations of flanking secondary structure. These results further demonstrate the energetic significance of local interactions in protein conformations. When used in combination with longer range energy functions, application of these potentials should lead to more accurate prediction of protein structure.

Computational Biology↗
PubMed2004

Direct demonstration of structural similarity between native and denatured eglin C.

To characterize the long-range structure that persists in the unfolded form of the 70-residue protein eglin C, residual dipolar couplings (RDCs) for HN-N and HA-CA bond vectors were measured by NMR spectroscopy for both its low pH, urea denatured state and its native state. When the data sets for the two different structural states were compared, a statistically significant correlation was found, with both sets of dipolar couplings yielding a correlation coefficient of r = 0.47 to 0.51. This finding directly demonstrates that the denatured state of eglin C has a nativelike global structure, a conclusion reached indirectly for staphylococcal nuclease by combining two different types of NMR data. A simple computer simulation showed that the degree of variation in phi and psi angles that yields the RDC correlation of r = 0.5 was inversely dependent on the statistical segment length, ranging from +/-6 to +/-30 degrees at the upper limit. Stable nativelike topologies that persist on unfolding would explain the rapid refolding kinetics displayed by many proteins and might provide a natural barrier against amyloid fibril formation.

Animals↗
PubMed2003

Observation of residual dipolar couplings in short peptides.

Residual dipolar couplings provide information on the orientation of individual bond vectors with respect to a unique set of molecular axes. We report that short peptides from 2 to 15 amino acids in length of arbitrary sequence exhibit a modest range of residual dipolar couplings when aligned in either strained polyacrylamide gels or alkyl-PEG bicelles. The absence of significant line broadening in gels suggests peptides align predominantly through steric interactions with the polyacrylamide matrix. However, broadening of NMR lines for a subset of residues aligned in bicelles indicates some peptides bind weakly to these lipid disks, yet a weak negative correlation between the couplings measured in gels and bicelles is consistent with steric hindrance playing a role in both media. The observation of dipolar couplings for peptides of length 10-15 suggests the statistical segment lengths of polypeptide chains must often be >10-15 residues, with data from denatured proteins indicating even larger values. Presumably, local side-chain backbone interactions severely restrict chain flexibility, with the cumulative effect of many such restrictions giving rise to biases in chain direction that may persist for the entire length of a protein chain. Comparison of experimental dipolar couplings for peptides with couplings calculated for ensembles of conformations generated by molecular dynamics should permit evaluation of the accuracy of molecular mechanics potentials in reproducing sequence-specific preferences for phi and psi angles.

Amino Acid Sequence↗
PubMed2003

Side chain dynamics in unfolded protein states: an NMR based 2H spin relaxation study of delta131delta.

NMR relaxation data on disordered proteins can provide insight into both structural and dynamic properties of these molecules. Because of chemical shift degeneracy in correlation spectra, detailed site-specific analyses of side chain dynamics have not been possible. Here, we present new experiments for the measurement of side chain dynamics in methyl-containing residues in unfolded protein states. The pulse schemes are similar to recently proposed methods for measuring deuterium spin relaxation rates in (13)CH(2)D methyl groups in folded proteins.(1) However, because resolution in (1)H-(13)C correlation maps of unfolded proteins is limiting, relaxation data are recorded as a series of (1)H-(15)N spectra. The methodology is illustrated with an application to the study of side chain dynamics in delta131delta, a large disordered fragment of staphylococcal nuclease containing residues 1-3 and 13-140 of the wide-type protein. A good correlation between the order parameters of the symmetry axes of the methyl groups and the backbone (1)H-(15)N bond vectors of the same residue is observed. Simulations establish that such a correlation is only possible if the unfolded state is comprised of an ensemble of structures which are not equiprobable. A motional model, which combines wobbling-in-a-cone and Gaussian axial fluctuations, is proposed to estimate chi(1) torsion angle fluctuations, sigma(chi)()1, of Val and Thr residues on the basis of the backbone and side chain order parameters. Values of sigma(chi)()1 are approximately 10 degrees larger than what has previously been observed in folded proteins. Of interest, the value of sigma(chi)()1 for Val 104 is considerably smaller than for other Val or Thr residues, suggesting that it may be part of a hydrophobic cluster. Notably large (15)N transverse relaxation rates are observed in this region. To our knowledge, this is the first time that side chain dynamics in an unfolded state have been studied in detail by NMR.

Deuterium↗
PubMed2003

Prediction of protein structure by emphasizing local side-chain/backbone interactions in ensembles of turn fragments.

The prediction strategy used in the CASP5 experiment was premised on the assumption that local side-chain/backbone interactions are the principal determinants of protein structure at low resolution. Our implementation of this assumption made extensive use of a scoring function based on the propensities of the 20 amino acids for 137 different sub-regions of the Ramachandran plot, allowing estimation of the quality of fit between a sequence segment and a known conformation. New folds were predicted in three steps: prediction of secondary structure, threading to isolate fragments of protein structures corresponding to one turn plus flanking helices/strands, and recombination of overlapping fragments. The most important step in this fragment ensemble approach, the isolation of turn fragments, employed 2 to 6 sequence homologues when available, with clustering of the best scoring fragments to recover the most common turn arrangement. Recombinants formed between 3 to 8 turn fragments, with cross-overs confined to helix/strand segments, were selected for compactness plus low energy as estimated by empirical amino acid pair potentials, and the most common overall topology identified by visual inspection. Because significant amounts of steric overlap were permitted during the recombination step, the final model was manually adjusted to reduce overlap and to enhance protein-like structural features. Even though only one or two models were submitted per target, for several targets the correct chain topology was predicted for fragment lengths up to 100 amino acids.

Binding Sites↗
PubMed2003

Effects of denaturants and substitutions of hydrophobic residues on backbone dynamics of denatured staphylococcal nuclease.

Analysis of residual dipolar couplings (RDCs) in the Delta131Delta fragment of staphylococcal nuclease has demonstrated that its ensemble-averaged structure is resistant to perturbations such as high concentrations of urea, low pH, and substitution of hydrophobic residues, suggesting that its residual structure is encoded by local side-chain/backbone interactions. In the present study, the effects of these same perturbations on the backbone dynamics of Delta131Delta were examined through (1)H-(15)N relaxation methods. Unlike the global structure reported by RDCs, the transverse relaxation rates R(2) were quite sensitive to denaturing conditions. At pH 5.2, Delta131Delta exhibits an uneven R(2) profile with several characteristic peaks involving hydrophobic chain segments. Protonation of carboxyl side chains by lowering the pH reduces the values of R(2) along the entire chain, yet these characteristic peaks remain. In contrast, high concentrations of urea or the substitution of 10 hydrophobic residues eliminates these peaks and reduces the R(2) values by a greater amount. The combination of low pH and high urea leads to further decreases in R(2). These denaturant-induced increases in backbone mobility are also reflected in decreases in (15)N NOEs and in relaxation interference parameters, with the former reporting an increase in fast motions and the latter a decrease in slow motions. Comparison between the changes in chain dynamics and the corresponding changes in Stokes radius and the patterns of RDCs suggests that regional variations in backbone dynamics in denatured nuclease arise primarily from local contacts between hydrophobic side chains and local interactions involving charged carboxyl groups.

Amino Acid Substitution↗
PubMed2003

Propensities, probabilities, and the Boltzmann hypothesis.

The relative strengths of interactions involving polypeptide chains can be estimated with reasonable accuracy with statistical potentials, free-energy functions derived from the frequency of occurrence of structural arrangements of residues or atoms in collections of protein structures. Recent published work has shown that the energetics of side-chain/backbone interactions can be modeled by the phi/psi propensities of the 20 amino acids. In this report, the more commonly used phi/psi probabilities are demonstrated to fail in evaluating the free energies of protein conformations because of an overriding preference for all helical structures. Comparison of the hypothetical reactions implied by these two different statistics-propensities versus probabilities-leads to the conclusion that the Boltzmann hypothesis may only be applicable for the calculation of statistical potentials after the starting conformation has been specified. This conclusion supports a simple conjecture: The surprising success of the Boltzmann hypothesis in explaining the energetics of protein structures is a direct consequence of a real equilibrium, one extending over evolutionary time that has maintained the stability of each protein within a narrow range of values.

Amino Acid Sequence↗
PubMed2002

Robustness of the long-range structure in denatured staphylococcal nuclease to changes in amino acid sequence.

A nativelike low-resolution structure has been shown to persist in the Delta 131 Delta denatured fragment of staphylococcal nuclease, even in the presence of 8 M urea. In this report, the physical-chemical basis of this structure is addressed by monitoring changes in structure reflected in residual dipolar couplings and diffusion coefficients as a function of changes in amino acid sequence. Ten large hydrophobic residues, previously shown to play dominant roles in the stability of the native state, are replaced with polar residues of similar shape. Modest increases in the Stokes radius determined by NMR methods result from replacement of five isoleucine/valine residues with threonine, one leucine with glutamine, and oxidation of four methionines to the sulfoxides. Yet in the presence of all ten hydrophobic to polar substitutions and 8 M urea, the NMR signature of a native-like topology is still largely intact. In addition, removal of 30 residues from either the N-terminus (which deletes a three-strand beta meander) or C-terminus (a long extended segment and the final alpha helix) produces only very small changes in long-range structure. These data indicate that both the general shape of the denatured state and the angular relationships of individual bond angles to the axes describing the spatial distribution of the protein chain are insensitive to large changes in the amino acid sequence, a finding consistent with the conclusion that the long-range structure of denatured proteins is encoded primarily by local steric interactions between side chains and the polypeptide backbone.

Binding Sites↗
PubMed2002

Molecular alignment of denatured states of staphylococcal nuclease with strained polyacrylamide gels and surfactant liquid crystalline phases.

Residual dipolar couplings reflect the orientation of vectors between pairs of magnetic nuclei relative to a unique set of molecular axes. Thus, unlike NOEs and scalar couplings, dipolar couplings provide access to long-range structural information. A prerequisite for measurement of these NMR parameters is imposition of a weak net alignment, most simply by forcing the macromolecules to tumble in an asymmetric environment that restricts some orientations more than others. In this report, several denatured forms of staphylococcal nuclease are aligned by using compressed and stretched polyacrylamide gels, a nonionic type of lipid bilayer disk or bicelle, and a liquid crystalline phase formed by a cationic lipid. All three types of media can be used at high urea concentrations. While polyacrylamide gels and bicelles produce similar alignment tensors through steric interactions, a liquid crystalline phase of cetylpyridinium bromide aligns denatured nuclease along a different set of axes, presumably through electrostatic effects. The analysis of residual dipolar couplings collected with two different alignment tensors may permit the calculation of ensembles of conformations. The dipolar couplings observed for staphylococcal nuclease denatured with urea, by low pH or by deletion of residues from both termini, suggest that all denatured forms share a common "topology", one which has been shown previously to be native-like. Although SDS/nuclease complexes give sharp and disperse (1)H-(15)N correlation spectra, only small couplings are observed in strained polyacrylamide gels.

Acids↗
PubMed2002

Composites of local structure propensities: evidence for local encoding of long-range structure.

To estimate how extensively the ensemble of denatured-state conformations is constrained by local side-chain-backbone interactions, propensities of each of the 20 amino acids to occur in mono- and dipeptides mapped to discrete regions of the Ramachandran map are computed from proteins of known structure. In addition, propensities are computed for the trans, gauche-, and gauche+ rotamers, with or without consideration of the values of phi and psi. These propensities are used in scoring functions for fragment threading, which estimates the energetic favorability of fragments of protein sequence to adopt the native conformation as opposed to hundreds of thousands of incorrect conformations. As finer subdivisions of the Ramachandran plot, neighboring residue phi/psi angles, and rotamers are incorporated, scoring functions become better at ranking the native conformation as the most favorable. With the best composite propensity function, the native structure can be distinguished from 300,000 incorrect structures for 71% of the 2130 arbitrary protein segments of length 40, 48% of 2247 segments of length 30, and 20% of 2368 segments of length 20. A majority of fragments of length 30-40 are estimated to be folded into the native conformation a substantial fraction of the time. These data suggest that the variations observed in amino acid frequencies in different phi/psi/chi1 environments in folded proteins reflect energetically important local side-chain-backbone interactions, interactions that may severely restrict the ensemble of conformations populated in the denatured state to a relatively small subset with nativelike structure.

Amino Acids↗