We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids. A completely synthetic gene was constructed that expresses a variant of the wild-type sequence with all 3 cysteines replaced with alanine. The resulting recombinant protein is active and folds reversibly. Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2-state process. Acid denaturation, however, reveals a cooperative transition at approximately pH 1.8 to a partially folded state. This acid state can be further denatured in a reversible 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 protein: the acid state displays a near-UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1-anilinonaphthalene, 8-sulfonate much more than either the native or unfolded states. Therefore, the acid state of E. coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core. Study holds ProTherm entries: 2763, 2764, 2765, 2766 Extra Details: pseudo wild type (all 3 cysteines have been replaced by Ala).,measurement was made in the presence of 0.5M GdnHCl ribonuclease HI; partially folded state; equilibrium unfolding;,Escherichia coli
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:19 p.m.
|Number of data points||7|
|Proteins||Ribonuclease HI ; Ribonuclease HI|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: dG_H2O ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV|