Electrophoretic characterization of the denatured states of staphylococcal nuclease

Thomas E. Creighton, David Shortle

Research output: Contribution to journalArticlepeer-review

Abstract

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 β-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).

Original languageEnglish (US)
Pages (from-to)670-682
Number of pages13
JournalJournal of molecular biology
Volume242
Issue number5
DOIs
StatePublished - Oct 6 1994

Keywords

  • Mutations
  • Protein stability
  • Staphylococcal nuclease
  • Unfolded proteins
  • Urea gradient electrophoresis

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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