High resistance of Escherichia coli ribonuclease HI variant with quintuple thermostabilizing mutations to thermal denaturation, acid denaturation, and proteolytic degradation.


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
Akasako A;Haruki M;Oobatake M;Kanaya S,Biochemistry (1995) High resistance of Escherichia coli ribonuclease HI variant with quintuple thermostabilizing mutations to thermal denaturation, acid denaturation, and proteolytic degradation. PMID:7794925
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ribonuclease HI A7ZHV1 RNH_ECO24
100.0 Ribonuclease HI B7MBJ0 RNH_ECO45
100.0 Ribonuclease HI P0A7Y6 RNH_ECO57
100.0 Ribonuclease HI B5Z0I8 RNH_ECO5E
100.0 Ribonuclease HI B7NKW4 RNH_ECO7I
100.0 Ribonuclease HI B7MQ23 RNH_ECO81
100.0 Ribonuclease HI B7M213 RNH_ECO8A
100.0 Ribonuclease HI C4ZRV1 RNH_ECOBW
100.0 Ribonuclease HI B1XD78 RNH_ECODH
100.0 Ribonuclease HI P0A7Y5 RNH_ECOL6
100.0 Ribonuclease HI B1IPU4 RNH_ECOLC
100.0 Ribonuclease HI P0A7Y4 RNH_ECOLI
100.0 Ribonuclease HI B7N876 RNH_ECOLU
100.0 Ribonuclease HI B6HZS7 RNH_ECOSE
100.0 Ribonuclease HI B1LHM3 RNH_ECOSM
100.0 Ribonuclease HI B7LW89 RNH_ESCF3
100.0 Ribonuclease HI B2U352 RNH_SHIB3
100.0 Ribonuclease HI Q325T2 RNH_SHIBS
100.0 Ribonuclease HI Q32JP9 RNH_SHIDS
100.0 Ribonuclease HI P0A7Y7 RNH_SHIFL
100.0 Ribonuclease HI Q3Z5E9 RNH_SHISS
99.4 Ribonuclease HI B7UJB0 RNH_ECO27
99.4 Ribonuclease HI B7LHC0 RNH_ECO55
99.4 Ribonuclease HI A7ZWF6 RNH_ECOHS
99.4 Ribonuclease HI Q0TLC3 RNH_ECOL5
93.5 Ribonuclease HI A8AKR0 RNH_CITK8
93.5 Ribonuclease HI B5F8X2 RNH_SALA4
93.5 Ribonuclease HI A9MPF1 RNH_SALAR
93.5 Ribonuclease HI Q57SZ6 RNH_SALCH
93.5 Ribonuclease HI B5FJ58 RNH_SALDC
93.5 Ribonuclease HI B5R449 RNH_SALEP
93.5 Ribonuclease HI B5R5L3 RNH_SALG2
93.5 Ribonuclease HI B4TK85 RNH_SALHS
93.5 Ribonuclease HI B4SV39 RNH_SALNS
93.5 Ribonuclease HI Q5PFD8 RNH_SALPA
93.5 Ribonuclease HI A9MZ19 RNH_SALPB
93.5 Ribonuclease HI B5BDW5 RNH_SALPK
93.5 Ribonuclease HI B4TYH0 RNH_SALSV
93.5 Ribonuclease HI P0A2C0 RNH_SALTI
93.5 Ribonuclease HI P0A2B9 RNH_SALTY
92.9 Ribonuclease HI C0Q6N2 RNH_SALPC
90.9 Ribonuclease HI B5Y1G2 RNH_KLEP3
90.9 Ribonuclease HI A6T512 RNH_KLEP7