Escherichia coli ribonuclease HI has a cavity within the hydrophobic core. Two core residues, Ala52 and Val74, resided at both ends of this cavity. We have constructed a series of single mutant proteins at Ala52, and double mutant proteins, in which Ala52 was replaced by Gly, Val, Ile, Leu, or Phe, and Val74 was replaced by Ala or Leu. All of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified. Measurement of the thermal denaturations of the proteins at pH 3.2 by CD suggests that the cavity is large enough to accommodate three methyl or methylene groups without creating serious strains. A correlation was observed between the protein stability and the hydrophobicity of the substituted residue. As a result, a number of the mutant proteins were more stable than the wild-type protein. The stabilities of the mutant proteins with charged or extremely bulky residues at the cavity were lower than those expected from the hydrophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins. However, examination of the far- and near-UV CD spectra and the enzymatic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein. These results suggest that the cavity in the hydrophobic core of E. coli RNase HI is conformationally fairly stable. This may be the reason why the cavity-filling mutations effectively increase the thermal stability of this protein. Study holds ProTherm entries: 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 13199, 13200, 13201, 13202, 13203, 13204, 13205, 13206, 13207, 13208, 13209, 13210, 13211, 13212, 13213, 13214, 13215, 13950, 13951, 13952, 13953, 13954, 13955, 13956, 13957, 13958 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:14 p.m.
|Number of data points||160|
|Proteins||Ribonuclease HI ; Ribonuclease HI|
|Assays/Quantities/Protocols||Experimental Assay: activity temp:53.0 C, ionic:-: - ; Experimental Assay: ddG ; Experimental Assay: activity ionic:: ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV|