ANION-INDUCED FOLDING OF STAPHYLOCOCCAL NUCLEASE - CHARACTERIZATION OF MULTIPLE EQUILIBRIUM PARTIALLY FOLDED INTERMEDIATES

The refolding of acid-unfolded staphylococcal nuclease (SNase) induced by anions was characterized, and revealed the existence of three diff erent partially folded intermediates (A states). The three intermediat es lack the rigid tertiary structure characteristic of native states, and differ in their degree of folding as measured by probes of seconda ry structure, size, stability and globularity. The least structured co nformation, A(1), is stabilized by chloride (600 mM) or sulfate (100 m M). It is about 50% folded (based on circular dichroism and small angl e X-ray scattering (SAXS) data). The next most structured intermediate , A(2), is induced by trifluor oacetate (300 mM) and has similar to 70 % native-like secondary structure. The most structured intermediate, A (3), is stabilized by trichloroacetate (50 mM) and has native-like sec ondary structure content and is almost as compact as the native state. The stability toward urea denaturation increases with increasing stru cture of the intermediates. Moreover, urea-induced unfolding studies s how that these partially folded species are separated from each other, and from the unfolded state, by significant free energy barriers, sug gesting that they are distinct conformational states. Kratky plots, ba sed on the SAXS data, indicate that the two more structured intermedia tes have significant globularity (i.e. a tightly packed core), whereas the less structured intermediate has very little globularity. These o bservations support a model of protein folding in which certain confor mations are of particularly low free energy and hence populated under conditions which differentially destabilize the native state. These pa rtially folded intermediates probably consist of ensembles of substate s with a common core of native-like secondary structure, which is resp onsible for their stability. Consequently, it is likely that the inter mediates observed here represent the equilibrium counterparts of trans ient kinetic intermediates. (C) 1998 Academic Press Limited.