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 rel ative hydrodynamic volumes as a function of urea concentration of stap h nuclease, and of mutant forms in which the native state mas substant ially destabilized, have been examined by urea gradient gel electropho resis. The native conformation is unique in its stabilization by speci fic ligands, which made it possible to distinguish between intermediat e 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 comp lex at high urea concentrations; plus two denatured states: (3) a part ly folded compact conformation that unfolded co-operatively and was se nsitive to mutation of residues in the beta-barrel portion of the prot ein, 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 ma y de due to the occurrence of the partly folded conformation (3).