Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes.
Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Biophysics, Yale University, New Haven, Connecticut 06511, USA.
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Wynn
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Anderson
F M
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Richards
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Fox
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D017510:Protein Folding; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D004220:Disulfides; D014508:Urea; D008836:Micrococcal Nuclease