Search PubMed⌕ Search

Biomedical subjects

D Shortle

Publications and source records attributed to D Shortle.

At least 37 records · Page 2Linked to original sources
PubMed1996

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics.

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.

Kinetics↗
PubMed1996

The denatured state (the other half of the folding equation) and its role in protein stability.

Experimental studies of the physical interactions that stabilize protein structure are complicated by the fact that proteins do not unfold to a simple reference state. When their folded structure breaks down, protein chains do not become random coils. Instead, they enter a poorly understood ensemble of partially folded states known collectively as the denatured state. Although it has long been held that agents that promote protein unfolding act specifically on the denatured state, the idea that mutations can exert their destabilizing (or in some cases, stabilizing) effects directly on this state is not widely accepted. A large body of thermodynamic data on mutant proteins plus a limited amount of structural information describing mutational effects on denatured states indicate that 1) the denatured state plays a central role in all aspects of protein stability, including mutant effects, and 2) a quantitative understanding of how amino acid sequence encodes protein structure will probably depend on a more complete picture of this complex, difficult-to-study state.

Mutation↗
PubMed1995

The equilibrium folding pathway of staphylococcal nuclease: identification of the most stable chain-chain interactions by NMR and CD spectroscopy.

In a previous report [Alexandrescu, A. T., Abeygunawardana, C., & Shortle, D. (1994) Biochemistry 33, 1063-1072], NMR methods were used to characterize the residual structure in delta 131 delta, a large fragment of staphylococcal nuclease that serves as a model denatured state under nondenaturing conditions. On the basis of a large number of missing amide protons for the residues that form a three-strand antiparallel beta sheet in the native state, it was concluded that this beta meander may be highly populated in delta 131 delta, with severe line broadening due to relatively slow exchange between different conformational states. In the present report, results from circular dichroism spectroscopy and NMR spectroscopy indicate strands beta 2-beta 3 form a beta hairpin at urea concentrations below 6 M. Amide proton resonances from several hydrophobic residues adjacent to this beta hairpin disappear in concert with all of the beta 2-beta 3 residues, suggesting a local, non-native hydrophobic interaction may help stabilize the beta hairpin. At concentrations below 3 M, all amide resonances from strand beta 1 in delta 131 delta also disappear, suggesting that beta 1 may combine with the beta 2-beta 3 hairpin to form a native-like beta meander. In addition, the hydrophobic helix alpha 2 decreases from approximately 30% population in 0 M urea to approximately 10%-15% at 6 M urea, whereas helix alpha 1 goes from 10%-15% populated in 0 M urea to undetectable in 6 M urea. Characterization of a second, distinctly different denatured state, WT nuclease at pH 3.0 and low salt, reveals that this low-density acid-denatured state is structurally similar to delta 131 delta at low concentrations of urea. From these and previously published data, a tenative equilibrium folding pathway can be constructed for staphylococcal nuclease which describes the relative strengths and interdependencies of the chain-chain interactions involved in forming the native state.

Amino Acid Sequence↗
PubMed1995

NMR structure of a stable "OB-fold" sub-domain isolated from staphylococcal nuclease.

Similar folds often occur in proteins with dissimilar sequences. The OB-fold forms a part of the structures of at least seven non-homologous proteins that share either oligonucleotide or oligosaccharide binding functions. A 1-103 fragment corresponding to the OB-fold of the 149 amino acid residue staphylococcal nuclease gives NMR spectra characteristic of an unfolded protein, i.e. the wild-type nuclease sequence is insufficient to maintain a stable tertiary structure in the absence of the C-terminal one-third of this single-domain protein. By contrast, the 1-103 fragment of nuclease with the mutations Val66Leu and Gly88Val adopts a stable tertiary structure. The NMR solution structure of this latter fragment is a close variation of the OB-fold found in the X-ray structure of the parent protein. The Val66Leu and Gly88Val mutations appear to stabilize tertiary structure by consolidating the hydrophobic core of the nuclease OB-fold sub-domain. Taken together, these results suggest that recurrent structural motifs such as the OB-fold may in some cases represent vestiges of autonomous folding units that, during evolution, have become integrated into more complex cooperative folding domains.

Magnetic Resonance Spectroscopy↗
PubMed1995

Initial studies of the equilibrium folding pathway of staphylococcal nuclease.

Spectroscopic methods were used to examine the sequential build up of structure in the denatured state of staphylococcal nuclease. The 'free energy distance' between the native and denatured states was manipulated by altering conditions in solution (for example altering urea or glycerol concentration) and by changing the amino acid sequences. Initial studies employed a fragment of nuclease, referred to as delta 131 delta, which lacks six structural residues from the amino terminus and one structural residue from the carboxy-terminus. Nuclear magnetic resonance analysis of this fragment in solution revealed a modest quantity of dynamic structure which is native-like in character. With the addition of urea, 12 new HN peaks appeared in the 1H-15N correlation spectrum, presumably as a result of the breakdown of residual structure involving the first three beta strands. With the addition of glycerol, there was a rapid increase in the quantity of beta sheet structure detected by circular dichroism spectroscopy. At very high glycerol concentrations, an increase in helical structure became apparent. These data in addition to previously published results suggest that: (i) a beta-meander (strands beta 1-beta 2-beta 3) and the second alpha helix (alpha 2) are among the most stable local structures; (ii) the five-strand beta-barrel forms in a reaction which does not require the presence of several other native substructures; and (iii) the last step on the equilibrium folding pathway may be the formation and packing of the carboxy terminal alpha helix (alpha 3) to give the native state.

Circular Dichroism↗
PubMed1995

The emerging role of insertions and deletions in protein engineering.

Most attempts to engineer the properties of proteins have employed single or multiple substitution mutations, which typically produce minor changes in structure. Recent structural and stability studies of insertion and deletion mutants clearly indicate that relatively large structural perturbations can be induced by altering the spacing of residues along the polypeptide backbone, often without major losses in protein stability. Although their effects are difficult to anticipate, insertions and deletions provide important new tools for altering protein structures in directions not achievable with substitutions alone.

Amino Acid Sequence↗
PubMed1994

Electrophoretic characterization of the denatured states of staphylococcal nuclease.

The denatured state of staphylococcal (staph) nuclease has been shown to be altered by mutations, which complicates studies of stability of the folded state and may be important for the folding process. The relative hydrodynamic volumes as a function of urea concentration of staph nuclease, and of mutant forms in which the native state was substantially destabilized, have been examined by urea gradient gel electrophoresis. The native conformation is unique in its stabilization by specific ligands, which made it possible to distinguish between intermediate electrophoretic mobilities resulting from a partly populated native conformation and from other partly folded conformations. Four distinct conformational states were observed; two native-like: (1) the native, with and without bound ligands, and (2) an altered native-ligand complex at high urea concentrations; plus two denatured states: (3) a partly folded compact conformation that unfolded co-operatively and was sensitive to mutation of residues in the beta-barrel portion of the protein, and (4) the unfolded state, which appeared from its hydrodynamic volume to be as unfolded as reduced, alkylated ribonuclease A, even at very low urea concentrations, and was not altered by single mutations. The effects of mutations on the denatured state of staph nuclease may be due to the occurrence of the partly folded conformation (3).

Calcium↗
PubMed1994

Backbone dynamics of a highly disordered 131 residue fragment of staphylococcal nuclease.

In order to characterize the dynamic properties of the denatured state of staphylococcal nuclease, R1, R2, and NOE relaxation parameters have been measured for the backbone 15N nuclei of a 131 residue fragment that serves as a model of the denatured state under non-denaturing conditions. The relaxation data indicate a wide range of amplitudes for segmental motion and are inconsistent with a random coil conformation. An optimal value of 7.8 ns was obtained for the molecular rotational correlation time tau m based on the analysis of the 79 residues for which R1, R2, and NOE relaxation data could be obtained. This value corresponds roughly to the slowest detectable motion on the nanosecond time scale and is of a magnitude consistent with global tumbling of a large portion of the molecule. For the majority of residues, experimental data could be described most adequately in terms of a modified "model-free" formalism which includes contributions from internal motions on both an intermediate (tau e) and a fast time scale (tau f) in the context of slow overall tumbling (tau m). The generalized order parameters S2, which gives the amplitude of motions on time scales faster than tau m, correlates with sequence hydrophobicity and suggests a relationship between chain flexibility and sequence propensity for hydrophobic collapse. The fractional populations of three alpha-helices in the protein show a stronger correlation with S2 values and hydrophobicities than with intrinsic helix propensities. These observations suggest that secondary structure may be preferentially stabilized in hydrophobic segments of the sequence.

Magnetic Resonance Spectroscopy↗
PubMed1994

Structure and dynamics of a denatured 131-residue fragment of staphylococcal nuclease: a heteronuclear NMR study.

A partially folded form of staphylococcal nuclease has been obtained by deleting residues 4-12 and 141-149 of the 149-residue wild-type protein. Sequence-specific NMR resonance assignments have been obtained for 106 of the 131 residues in this protein fragment by using multi-dimensional triple resonance NMR of samples enriched with 13C and 15N. Residues corresponding to helix 2 (residues 98-106) and helix 1 (residues 54-68) of the native state give chemical shifts and NOE effects characteristic of helical structure. These same residues, however, give coupling constants and NOE effects indicative of fast conformational averaging between helical and extended conformations. The residual helix structure observed in the nuclease fragment is thus considerably less persistent than the corresponding structure in the native state. Based on H alpha chemical shifts, we estimate the fractional population of helical conformers to be 30% for helix 2 and 10% for helix 1. Two segments, 83-86 and 94-97, show NOE effects, coupling constants, and lowered amide temperature coefficients consistent with a native-like reverse-turn structure. The C-terminal alpha-helix as well as the fourth and fifth strands of the 5-strand beta-barrel show little evidence for ordered structure. The first three strands of the beta-sheet, part of the catalytic loop, and the first turn of helix 3 give significantly poorer NMR data than the rest of the protein, possibly as a result of exchange broadening, and could not be characterized in detail. That the most persistent elements of structure in the fragment are native-like suggests that nuclease may fold by a hierarchical mechanism.

Amides↗
PubMed1994

Evidence for strained interactions between side-chains and the polypeptide backbone.

In most proteins, a small but significant fraction of residues adopt phi,psi angles that generate unfavorable steric interactions between side-chain atoms and the peptide backbone. For the small protein staphylococcal nuclease, the X-ray structure reveals that 18 of 133 residues occupy unusual and, presumably, energetically unfavorable backbone conformations. To quantify the amount of strain energy generated by these local interactions, we have analyzed the changes in stability that accompany replacement of the wild-type side-chain with glycine, a residue that can access a much larger set of phi,psi angles without energy penalty. To correct for the many other sources of stability loss that might accompany this mutation, the glycine mutant was compared to an alanine mutant at the same position and the resulting free energy difference delta delta GG-->A was then compared to the average delta delta GG-->A at all other, unstrained positions in the nuclease occupied by similar amino acid types. In addition, potential steric clashes were introduced by substituting alanine at each of six positions occupied in the wild-type by glycine with phi,psi angles that are unfavorable for all other residue types. The data suggest that residues with phi,psi angles outside the preferred alpha-helical and beta-sheet regions represent sites of local strain energy that lower the stability of the native state by 1 to 2 kcal/mol and, in some cases, as much as 3 to 4 kcal/mol. Given that 10 to 20% of residues in globular proteins adopt phi,psi angles outside the preferred alpha-helical and beta-sheet regions, this implies that there is on the order of 20 kcal/mol of strain energy in a protein of 100 residues that may be relieved by appropriate mutations.

Amino Acid Sequence↗
PubMed1993

NMR analysis of the residual structure in the denatured state of an unusual mutant of staphylococcal nuclease.

BACKGROUND: Staphylococcal nuclease is a well-developed model system for analyzing the effects of mutations on protein folding and stability. Substitution of glycine 88 with valine (Gly88Val) destabilizes staphylococcal nuclease by 1.0 kcal mole-1 and reduces its sensitivity to the denaturant guanidine hydrochloride, a phenomenon which may indicate an increase in residual structure in the denatured state. To assess its effects on denatured state structure, the Gly88Val mutation was incorporated into a 136 residue nonsense fragment which has been developed as a model of the wild type denatured state. RESULTS: Application of two- and three-dimensional NMR spectroscopy to the Gly88Val fragment uniformly labeled with 15N and 13C has led to the assignment of 93 of the 136 residues. Comparison of chemical shifts of backbone resonances to those of wild type native nuclease, analysis of the secondary shifts of the assigned resonances and nuclear Overhauser effects involving backbone protons indicate that, unlike the wild type fragment, most if not all of the five-stranded beta-barrel structure persists in this denatured state. CONCLUSION: One major effect of the Gly88Val mutation is to perturb the cooperative breakdown of the folded conformation, leading to a denatured state which is both more ordered and more stable than that formed by the wild type sequence. Since the equilibrium between the native and denatured states depends on the free energy difference between them, stabilization of the denatured state by the Gly88Val mutation indirectly destabilizes the native state.

Amino Acid Sequence↗
PubMed1993

Patterns of nonadditivity between pairs of stability mutations in staphylococcal nuclease.

To identify interactions between amino acid positions in staphylococcal nuclease that affect its stability, a collection of 71 double-mutant forms was constructed from 22 previously characterized single mutants. These single mutations were assigned to three different classes on the basis of their m value [m = d(delta G)/d[GuHCl]], a parameter that has been correlated with energetically significant changes in the structure of the denatured state [Green et al. (1992) Biochemistry 31,5717-5728]. Several mutant pairs from five of the six possible double-mutant classes were analyzed by guanidine hydrochloride denaturation to determine the extent to which changes in stability (delta delta GH2O) and changes in the m value (delta mGuHCl) reflect the sum of the effects of the individual mutants. The differences between the values for delta delta GH2O and delta mGuHCl estimated on the assumption of additivity and those obtained by experiment, i.e., delta delta delta G and delta delta m, were calculated for each double-mutant protein. Surprisingly, a large majority of double mutants from four of the five classes exhibited positive values of delta delta delta G and delta delta m; i.e., they were more stable and displayed a higher sensitivity to GuHCl than predicted on the basis of additivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The alpha aneurism: a structural motif revealed in an insertion mutant of staphylococcal nuclease.

The x-ray crystal structure of a mutant of staphylococcal nuclease that contains a single glycine residue inserted in the C-terminal alpha-helix has been solved to 1.67 A resolution and refined to a crystallographic R value of 0.170. This inserted glycine residue is accommodated in the alpha-helix by formation of a previously uncharacterized bulge, which we term the alpha aneurism. A conformational search of known protein structures has identified the alpha aneurism in a number of protein families, including the histocompatibility antigens and hemoglobins.

Amino Acid Sequence↗