Thermal stabilities of mutant ribonuclease HI proteins from Escherichia coli, in which each of five histidine residues was replaced with alanine, were examined at various pHs. Increases in the Tm values were observed at pH 3.0 for four of the mutant proteins, in which each of the four histidine residues exposed to the solvent was mutated, as compared to the Tm of the wild-type protein. The thermostabilization of three of the mutant proteins was dependent on pH, and only observed at low pH. The thermostabilizing effects of the His-->Ala substitutions were cumulative. The temperature of the midpoint of the transition in the thermal unfolding curves, Tm, of the most stable mutant enzyme, in which His 62, His 83, His 124, and His 127 were replaced by Ala, was 5.5 degrees C higher than that of the wild-type enzyme at pH 3.0. The stability of the wild-type protein decreased as the pH was lowered below pH 4, a condition favoring the protonation of carboxyl groups, probably due to unfavorable electrostatic interactions introduced by the increase in positive charges on the protein. Since imidazole groups are positively charged at pH 3.0, it seems likely that thermal stabilization at pH 3.0 by a His-->Ala substitution would be the result of a reduction in such unfavorable electrostatic interactions. These results suggest that amino acid substitutions that cause a decrease in the number of positive charges on the surface of a protein can be used as a general strategy to enhance protein stability at pH values below pH 4. Study holds ProTherm entries: 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731 Extra Details: Escherichia coli ribonuclease HI; thermal stability;,protein stability; thermal unfolding
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:15 p.m.
|Number of data points||70|
|Proteins||Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI|
|Assays/Quantities/Protocols||Experimental Assay: Cm buffers:Tris-HCl: 20 mM, pH:8.2 ; Experimental Assay: dG_H2O buffers:Tris-HCl: 20 mM, pH:8.2 ; Experimental Assay: Cm pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: dG_H2O pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: Tm buffers:Sodium phosphate: 20 mM, pH:7.8 ; Experimental Assay: Tm pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: Tm pH:3.0, buffers:glycine-HCl: 10 mM ; Derived Quantity: ddG_H2O buffers:Tris-HCl: 20 mM, pH:8.2 ; Derived Quantity: ddG_H2O pH:5.5, buffers:Sodium acetate: 20 mM ; Derived Quantity: dTm buffers:Sodium phosphate: 20 mM, pH:7.8 ; Derived Quantity: dTm pH:5.5, buffers:Sodium acetate: 20 mM ; Derived Quantity: dTm pH:3.0, buffers:glycine-HCl: 10 mM|
|Libraries||Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|