CHARACTERIZATION OF THE STABLE, ACID-INDUCED, MOLTEN GLOBULE-LIKE STATE OF STAPHYLOCOCCAL NUCLEASE

Titration of a salt-free solution of native staphylococcal nuclease by HCl leads to an unfolding transition in the vicinity of pH 4, as dete rmined by near- and far-UV circular dichroism. At pH 2-3, the protein is substantially unfolded. The addition of further HCl results in a se cond transition, this one to a more structured species (the A state) w ith the properties of an expanded molten globule, namely substantial s econdary structure, little or no tertiary structure, relatively compac t size as determined by hydrodynamic radius, and the ability to bind t he hydrophobic dye 1-anilino-8-naphthalene sulfonic acid. The addition of anions, in the form of neutral salts, to the acid-unfolded state a t pH 2 also causes a transition leading to the A state. Fourier transf orm infrared analysis of the amide I band was used to compare the amou nt and type of secondary structure in the native and A states. A signi ficant decrease in alpha-helix structure, with a corresponding increas e in beta or extended structure, was observed in the A state, compared to the native state. A model to account for such compact denatured st ates is proposed.