Thermodynamics of staphylococcal nuclease denaturation. II. The A-state.
Staphylococcal nuclease, at low pH and in the presence of high salt concentrations, has previously been proposed to exist in a partially folded 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 at pH 2.1 in the presence of moderate to high salt concentrations and at low temperature exists in a substantially folded form structurally more similar to a native state. The A-state has the far-UV circular dichroism spectra characteristic of the native protein, which indicates that it has a large degree of secondary structure. Upon heating, the A-state denatures with a sigmoidal change in far-UV ellipticity and an observable peak in a differential scanning calorimeter trace, indicating that it is thermodynamically distinct from the denatured state. Three different mutations in a residue normally buried in the protein's core stabilize or destabilize the A-state in the same way as they affect the denaturation of the native state. The A-state must, therefore, contain 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.
Carra
J H
JH
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Anderson
E A
EA
Privalov
P L
PL
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008836:Micrococcal Nuclease / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D008836:Micrococcal Nuclease