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.


CONCLUSIONS
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.
Shortle
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Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Abeygunawardana
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D000595:Amino Acid Sequence; D002151:Calorimetry; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D005998:Glycine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008836:Micrococcal Nuclease / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014633:Valine
D011994:Recombinant Proteins; D008836:Micrococcal Nuclease; D014633:Valine; D005998:Glycine