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D Shortle

Publications and source records attributed to D Shortle.

At least 19 recordsLinked to original sources
PubMed2001

Persistence of native-like topology in a denatured protein in 8 M urea.

Experimental methods have demonstrated that when a protein unfolds, not all of its structure is lost. Here we report measurement of residual dipolar couplings in denatured forms of the small protein staphylococcal nuclease oriented in strained polyacrylamide gels. A highly significant correlation among the dipolar couplings for individual residues suggests that a native-like spatial positioning and orientation of chain segments (topology) persists to concentrations of at least 8 molar urea. These data demonstrate that long-range ordering can occur well before a folding protein attains a compact conformation, a conclusion not anticipated by any of the standard models of protein folding.

Acrylic Resins↗
PubMed2000

Correlation between changes in nuclear magnetic resonance order parameters and conformational entropy: molecular dynamics simulations of native and denatured staphylococcal nuclease.

Recent work has suggested that changes in NMR order parameters may quantitatively reflect changes in the conformational entropy of a protein ensemble. The extent of the mathematical relationship between local entropy changes as seen by NMR order parameters and the full protein entropy change is a complex issue. As a step towards a fuller understanding of this problem, molecular dynamics calculations of both native and denatured staphylococcal nuclease were performed. The N-H bond vector motion, in both explicit and implicit solvent, was analyzed to estimate local and global entropy changes. The calculated N-H bond vector order parameters from simulation agreed on average with experimental values for both native and denatured structures. However, the inverted-U profile of order parameters versus residue number observed experimentally for denatured nuclease was only partially reproduced by simulation of compact denatured structures. Comparisons made across the full set of simulations revealed a correlation between the N-H order parameter-based conformational entropy change and the total quasiharmonic-based conformational entropy change between the native and denatured structures. The calculations showed that about 25% of the total entropy change was reflected by changes in simulated S2 values. This result suggests that NMR-derived order parameters may be used to provide a reasonable estimate of the total conformational entropy change on protein folding.

Magnetic Resonance Spectroscopy↗
PubMed1999

Structure prediction: The state of the art.

A recent meeting to evaluate the state of the art of protein structure prediction saw progress on all fronts; for prediction methods based on comparative modeling or fold recognition, the progress was incremental, but in the case of ab initio structure prediction, some surprising successes were reported.

Algorithms↗
PubMed1999

A model of the changes in denatured state structure underlying m value effects in staphylococcal nuclease.

Hydrogen exchange kinetics were measured on the native states of wild type staphylococcal nuclease and four mutants with values of mGuHCl (defined as dDeltaG/d[guanidine hydrochloride]) ranging from 0.8 to 1.4 of the wild type value. Residues within the five-strand beta-barrel of wild type and E75A and D77A, two mutants with reduced values of m GuHCl, were significantly more protected from exchange than expected on the basis of global stability as measured by fluorescence. In contrast, mutants V23A and M26G with elevated values of mGuHCl approach a flat profile of more or less constant protection independent of position in the structure. Differences in exchange protection between the C-terminus and the beta-barrel region correlate with mGuHCl, suggesting that a residual barrel-like structure becomes more highly populated in the denatured states of m- mutants and less populated in m+ mutants. Variations in the population of such a molten globule-like structure would account for the large changes in solvent accessible surface area of the denatured state thought to underlie m value effects.

Amino Acid Substitution↗
PubMed1999

Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.

Amino Acid Sequence↗
PubMed1998

Clustering of low-energy conformations near the native structures of small proteins.

Recent experimental studies of the denatured state and theoretical analyses of the folding landscape suggest that there are a large multiplicity of low-energy, partially folded conformations near the native state. In this report, we describe a strategy for predicting protein structure based on the working hypothesis that there are a greater number of low-energy conformations surrounding the correct fold than there are surrounding low-energy incorrect folds. To test this idea, 12 ensembles of 500 to 1,000 low-energy structures for 10 small proteins were analyzed by calculating the rms deviation of the Calpha coordinates between each conformation and every other conformation in the ensemble. In all 12 cases, the conformation with the greatest number of conformations within 4-A rms deviation was closer to the native structure than were the majority of conformations in the ensemble, and in most cases it was among the closest 1 to 5%. These results suggest that, to fold efficiently and retain robustness to changes in amino acid sequence, proteins may have evolved a native structure situated within a broad basin of low-energy conformations, a feature which could facilitate the prediction of protein structure at low resolution.

Computer Simulation↗
PubMed1997

Comprehensive NOE characterization of a partially folded large fragment of staphylococcal nuclease Delta131Delta, using NMR methods with improved resolution.

Comprehensive NOE results from detailed structural characterization of a 131 residue partially folded fragment of staphylococcal nuclease (Delta131Delta) made possible by NMR methods with improved resolution are presented. The resulting NOE patterns reflect sampling of both alpha and beta regions of phi, phi conformational space, yet demonstrate significant preferences for both native-like and non-native-like turn and potentially helical conformations. Together with data from studies of the unfolded state of the drkN SH3 domain, NOE patterns observed for partially folded or unfolded proteins are summarized. It is surprising that few long-range NOEs were observed in Delta131Delta. The two longest-range NOEs are both native-like; one of these, an (i,i+5) NOE, provides evidence for a Schellman capping motif for helix termination. Many aliphatic-aliphatic and aliphatic-amide NOEs, which are not normally observed in folded proteins, were detected. We have ruled out significant contributions from spin-diffusion for a number of these NOEs and suggest that one source may be sampling of non-prolyl cis peptide bond configurations in the disordered state of Delta131Delta.

Amino Acid Sequence↗
PubMed1997

Characterization of long-range structure in the denatured state of staphylococcal nuclease. II. Distance restraints from paramagnetic relaxation and calculation of an ensemble of structures.

Structural analysis of delta131delta, a fragment model of the denatured state of staphylococcal nuclease, has been extended by obtaining long-range distance restraints between chain segments by paramagnetic relaxation enhancement. Fourteen unique PROXYL spin labels were introduced at sites that are solvent-exposed in the native state, and the resulting enhancements of T2 for the amide protons were measured by NMR spectroscopy. When these data were combined with either measured or estimated correlation times tau(c), the r(-6)-weighted, time and ensemble-averaged distance between the spin label and 30 to 60 amide protons could be calculated for each spin-labeled protein. On the basis of approximately 700 such loose distance restraints, ensembles of compatible structures were generated by a combined distance geometry/molecular dynamics approach. Because of the large uncertainty in the physical basis of these distance restraints, a number of calculations were carried out to establish the sensitivity of the calculated structures to systematic errors in these restraints. Overall, the structural features reflected in the paramagnetic relaxation data were robust; large variations in tau(c), in the bounds window of allowed distances, or in the number of restraint distances used had small effects on the general features common to all calculated structures. The global topology of this denatured form of staphylococcal nuclease, as described by an ensemble of conformations consistent with the data, is strikingly similar to that of the native state, the major difference being the segregation of two hydrophobic segments that form a beta hairpin in the native state. These findings suggest that the topology of a protein's fold is established in the denatured state in the absence of cooperative interactions involving tight packing or stable hydrogen bonding. Hydrophobic interactions alone may encode global topology.

Algorithms↗
PubMed1997

Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. Paramagnetic relaxation enhancement by nitroxide spin labels.

Structural analysis of delta131delta, a fragment model of the denatured state of staphylococcal nuclease, has been extended by obtaining long-range distance restraints between protein chain segments based on paramagnetic relaxation enhancement methods. PROXYL spin labels were attached at unique cysteine residues introduced at 14 different sites along the polypeptide chain, and the resulting enhancements of amide proton relaxation were measured by NMR spectroscopy. To minimize perturbation of denatured state structure, these labeling sites were chosen on the basis of a high solvent exposure in the native state and a small change in stability and m-value upon mutation of the wild-type residue to cysteine or alanine. EPR spectroscopy confirmed that in all cases the PROXYL label of the modified protein was solvent-exposed and undergoing free isotropic rotation. By quantifying at 500 MHz and 600 MHz the enhancement of both T1 and T2 relaxation for amide protons resolved in a 1H-15N correlation spectrum, the apparent correlation time for the free electron-proton vectors for six PROXYL-labeled proteins could be estimated. With these data plus the enhancements in transverse relaxation rate (R2) for the other eight proteins, the time-averaged, r(-6) weighted distance between the free electron on the unique nitroxide and 30 to 60 amide protons in each protein could be approximated. Inspection of the pattern of R2 enhancements reveals a significant amount of long-range structure in this denatured state, a clear indication that it is not a random coil.

Amides↗
PubMed1997

Structure prediction: folding proteins by pattern recognition.

Although we are still a long way from being able to predict the details of protein structure from the underlying chemistry, slow but steady progress is being made at modeling structural features by recognizing the patterns that connect sequence to structure.

Animals↗
PubMed1997

Residual helical and turn structure in the denatured state of staphylococcal nuclease: analysis of peptide fragments.

BACKGROUND: Previous NMR studies of the denatured state of staphylococcal nuclease identified three significantly populated native-like secondary structures: the second alpha-helix, alpha 2 (residues 98-106), estimated to be present 30% of the time, and two highly populated beta-turns, a type I turn (residues 83-86) and a type I' turn (residues 94-97). In the absence of detectable beta-structure or long-range interactions in this low-density denatured state, these three secondary structures appeared to be stabilized exclusively by local interactions. RESULTS: To quantitate the intrinsic stability of these secondary structures, three synthetic peptides corresponding in sequence to these chain segments, plus 2-4 flanking residues, have been analyzed by NMR and CD spectroscopy. Neither of the turn peptides showed significant evidence of residual structure. The data for the alpha 2 peptide suggest that this alpha-helix remains approximately 30% helical when separated from the rest of the protein. However, the type I' turn and the Schellman motif, at the amino and carboxyl termini, respectively, observed in both the native and the denatured state, do not form in this peptide. Instead, the helix appears to propagate to the ends of the peptide, overriding both of these helix-stop signals. CONCLUSIONS: The presence of a native-like secondary structure in a denatured protein does not necessarily imply that it has a high intrinsic stability. beta-turns in particular can be stabilized by long-range interactions in the absence of stable beta-strands. In addition, so-called helix-stop signals, such as the Schellman motif, may not contribute actively to helix stability. As for turns, these local interactions at the ends of helices may be passive structures that form in response to longer-range interactions.

Amino Acid Sequence↗
PubMed1997

Triple-resonance NOESY-based experiments with improved spectral resolution: applications to structural characterization of unfolded, partially folded and folded proteins.

NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C' (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C' resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in 1H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C' either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), delta 131 delta, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate H alpha protons or protons resonating close to water.

Amino Acid Sequence↗
PubMed1997

Measurement of water-amide proton exchange rates in the denatured state of staphylococcal nuclease by a magnetization transfer technique.

The rates of hydrogen exchange were measured in a "physiological" denatured state of staphylococcal nuclease using a NMR magnetization transfer experiment suitable for the measurement of exchange rates faster than 0.5 s-1. The results are compared with predicted exchange rates (kex) for the random coil state (Bai et al., Proteins 17:75-86, 1993). No protection factors (= predicted rate/measured rate) larger than 2.4 were observed, consistent with other NMR data which strongly suggest only small amounts of residual secondary structure in this denatured state. Systematically low protection factors (0.51 +/- 0.23) were found for Asp and Glu residues, while high protection factors were observed for Gly (1.60 +/- 0.60). We conclude that the predicted exchange rates (kex) may have an uncertainty of 2- to 3-fold. Thus, for denatured proteins only protection factors with a value of 5 or larger can be assigned structural significance. These results also demonstrate that multidimensional magnetization transfer NMR techniques are powerful tools in this research field due to its ability to measure rapidly exchanging protons (> 05 s-1) with high accuracy.

Amides↗
PubMed1996

Contributions of the ionizable amino acids to the stability of staphylococcal nuclease.

To quantitate the contributions of the ionizable amino acids to the stability of the native state of staphylococcal nuclease, each of the 23 lysines, 5 arginines, 4 histidines, 12 glutamic acids, and 8 aspartic acids was substituted with both alanine and glycine. This collection of 104 mutant proteins was analyzed by guanidine hydrochloride (GuHCl) denaturation, using intrinsic tryptophan fluorescence to quantitate the equilibrium between native and denatured states. From the analysis of these data, each mutant protein's stability in the absence of denaturant (delta GH2O) and sensitivity to changes in denaturant concentration [mGuHCl = d(delta G)/d[GuHCl]] were obtained. Several general trends in these values suggest that electrostatic interactions make only a minor contribution to the net stability of this protein. For the residue pairs that form ten salt bridges and ten charged hydrogen bonds between side chains, no correlation was observed between the stability losses (delta delta G) accompanying alanine substitution of each member of the pair. Little or no significant correlation was found between the magnitude of the loss in stability and the local electrostatic potential calculated from the three-dimensional structure by numerical and model dependent solutions of the linearized Poisson-Boltzmann equation. The structural parameters which correlated most strongly with stability loss are measures of the extent of burial of the residue in the native structure, as was previously observed for alanine and glycine substitutions of large hydrophobic residues [Shortle et al. (1990) Biochemistry 29, 8033] and of the polar, uncharged residues [Green et al. (1992) Biochemistry 31, 5717]. These results suggest that the ionizable amino acids contribute to stability predominantly through packing and bonding interactions that do not depend on their electrostatic charge.

Amino Acids↗
PubMed1996

Protein folding for realists: a timeless phenomenon.

Future research on protein folding must confront two serious dilemmas. (1) It may never be possible to observe at high resolution the very important structures that form in the first few milliseconds of the refolding reaction. (2) The energy functions used to predict structure from sequence will always be approximations of the true energy function. One strategy to resolve both dilemmas is to view protein folding from a different perspective, one that no longer emphasizes time and unique trajectories through conformation space. Instead, free energy replaces time as the reaction coordinate, and ensembles of equilibrium states of partially folded proteins are analyzed in place of trajectories of one protein chain through conformation space, either in vitro or in silico. Initial characterization of the folding of staphylococcal nuclease within this alternative conceptual framework has led to an equilibrium folding pathway with several surprising features. In addition to the finding of two bundles of four hydrophobic segments containing both native and non-native interactions, a gradient in relative stability of different substructures has been identified, with the most stable interactions located toward the amino terminus and the least stable toward the carboxy terminus. Hydrophobic bundles with up-down topology and stability gradients may be two examples of numerous tactics used by proteins to facilitate rapid folding and minimize aggregation. As NMR methods for structural analysis of partially folded proteins are refined, higher resolution descriptions of the structure and dynamics of the polypeptide chain outside the native state may provide many insights into the processes and energetics underlying the self-assembly of folded structure.

Forecasting↗
PubMed1996

A dynamic bundle of four adjacent hydrophobic segments in the denatured state of staphylococcal nuclease.

In an earlier study of the denatured state of staphylococcal nuclease (Wang Y, Shortle D, 1995, Biochemistry 34:15895-15905), we reported evidence of a three-strand antiparallel beta sheet that persists at high urea concentrations and is stabilized by a local "non-native" interaction with four large hydrophobic residues. Because the amide proton resonances for all of the involved residues are severely broadened, this unusual structure is not amenable to conventional NMR analysis and must be studied by indirect methods. In this report, we present data that confirm the important role of interactions involving four hydrophobic residues (Leu 36, Leu 37, Leu 38, and Val 39) in stabilizing the structure formed by the chain segments corresponding to beta 1-beta 2-beta 3-h, interactions that are not present in the native state. Glycine substitutions for each of these large hydrophobic residues destabilizes or disrupts this beta structure, as assessed by HN line sharpening and changes in the CD spectrum. The 13C resonances of the carbonyl carbon for several of the residues in this structure indicate conformational dynamics that respond in a complex way to addition of urea or changes in sequence. Studies of hydrogen exchange kinetics in a closely related variant of staphylococcal nuclease demonstrate the absence of the stable hydrogen bonding between the strands expected for a native-like three-strand beta sheet. Instead, the data are more consistent with the three beta strand segments plus the four adjacent hydrophobic residues forming a dynamic, aligned array or bundle held together by hydrophobic interactions.

Carbon Isotopes↗