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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.