Abstract

To test whether the combination of multiple thermostabilizing mutations is a useful strategy to generate a hyperstable mutant protein, five mutations, Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His or Asn, were simultaneously introduced into Escherichia coli ribonuclease HI. The enzymatic activities of the resultant quintuple mutant proteins, 5H- and 5N-RNases HI, which have His and Asn at position 134, respectively, were 35 and 55% of that of the wild-type protein. The far-UV and near-UV CD spectra of these mutant proteins were similar to those of the wild-type protein, suggesting that the mutations did not seriously affect the tertiary structure of the protein. The differences in the free energy change of unfolding between the wild-type and mutant proteins, delta delta G, were estimated by analyzing the thermal denaturation of the proteins by CD. The 5H-RNase HI protein, which was slightly more stable than the 5N-RNase HI, was more stable than the wild-type protein by 20.2 degrees C in Tm and 5.6 kcal/mol in delta G at pH 5.5. In addition, the 5H-RNase HI was highly resistant to proteolysis and acid denaturation. The effects of each mutation on the thermal stability and the susceptibility to chymotryptic digestion were nearly cumulative, and the 5H-RNase HI undergoes chymotryptic digestion at a rate that is 41 times slower than that of the wild-type protein. Good correlation was observed between the thermal stability and the resistance to chymotryptic digestion for all proteins examined.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 13343, 13344, 13345, 13346, 13347, 13348, 13349, 13350 Extra Details: Escherichia coli ribonuclease HI; thermal denaturation;,enzymatic activities; tertiary structure; thermal stability

Submission Details

ID: i8uNNj6U4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details

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