EQUILIBRIUM UNFOLDING OF ESCHERICHIA-COLI RIBONUCLEASE-H - CHARACTERIZATION OF A PARTIALLY FOLDED STATE

We have examined the equilibrium unfolding of Escherichia coli ribonuc lease HI (RNase H), a member of a family of enzymes that cleaves RNA f rom RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild-type sequence with all 3 cysteines rep laced with alanine. The resulting recombinant protein is active and fo lds reversibly. Denaturation studies monitored by circular dichroism a nd tryptophan fluorescence yield coincident curves that suggest the eq uilibrium unfolding reaction is a 2-state process. Acid denaturation, however, reveals a cooperative transition at similar to pH 1.8 to a pa rtially folded state. This acid state can be further denatured in a re versible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence. Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric . Although compact, the acid state does not resemble the native protei n: the acid state displays a near-UV CD spectrum similar to the unfold ed state and binds to and enhances the fluorescence of the dye 1-anili nonaphthalene, 8-sulfonate much more than either the native or unfolde d states. Therefore, the acid state of E. coli RNase H has the charact eristics of a molten globule: it retains a high degree of secondary st ructure, remains compact, yet does not appear to contain a tightly pac ked core.