Effect of proline mutations on the stability and kinetics of folding of staphylococcal nuclease.
The role of proline in the stability and kinetics of folding of wild-type staphylococcal nuclease and its P117G, P117T, and P31A mutants was examined as a function of guanidinium thiocyanate (Gdn-SCN) concentration. Replacement of Pro-117 with Gly or Thr caused small increases in stability, whereas substitution of Pro-31 by Ala led to a small decrease in stability. The slopes of the plots of delta G against denaturant concentration (m) for the mutant proteins are significantly smaller than for the wild-type, suggesting a decrease in the solvent-accessible surface area of the denatured state relative to that of the wild-type. The rates of unfolding and refolding were monitored using tryptophan fluorescence. The kinetic traces for refolding in the presence of Gdn-SCN were triphasic for the wild-type protein and P31A but biphasic for P117G and P117T mutants. The slower phases were typically 10% of the total amplitude except in the transition region. The rates of the fastest and medium phases of the wild-type were essentially unaffected by the mutations. Double-jump experiments in which the protein was unfolded in a high concentration of denaturant for a short time period and then refolded to final Gdn-SCN concentrations near the Cm revealed a fast increase in fluorescence emission corresponding to formation of the native state, followed by a slower decrease with an amplitude that varied with the guanidine concentration and time of unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)
Nakano
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Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064.
Antonino
L C
LC
Fox
R O
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Fink
A L
AL
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D011392:Proline; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D011994:Recombinant Proteins; D011392:Proline; D008836:Micrococcal Nuclease; D019791:Guanidine