THERMODYNAMICS OF STAPHYLOCOCCAL NUCLEASE DENATURATION .2. THE A-STATE

Staphylococcal nuclease, at low pH and in the presence of high salt co ncentrations, has previously been proposed to exist in a partially fol ded or molten globule form called the ''A-state'' (Fink et al., 1993, Protein Sci 2:1155-1160). We have found that the A-state of nuclease a t pH 2.1 in the presence of moderate to high salt concentrations and a t low temperature exists in a substantially folded form structurally m ore similar to a native state. The A-state has the far-UV circular dic hroism spectra characteristic of the native protein, which indicates t hat it has a large degree of secondary structure. Upon heating, the A- state denatures with a sigmoidal change in far-UV ellipticity and an o bservable peak in a differential scanning calorimeter trace, indicatin g that it is thermodynamically distinct from the denatured state. Thre e different mutations in a residue normally buried in the protein's co re stabilize or destabilize the A-state in the same way as they affect the denaturation of the native state. The A-state must, therefore, co ntain at least some tertiary packing of side chains. Unlike the native state, which shows cold denaturation at low temperatures, the A-state is most stable at temperatures below 0 degrees C.