Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution.


The insertion of a Gly residue (designated as Gly-80b) between the C-cap of the alpha II-helix (Gln-80) and the N-cap of the alpha III-helix (Trp-81) in Escherichia coli ribonuclease HI enhances the protein stability by 0.4 kcal/mol in delta G (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., & Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Another mutation within the alpha II-helix, Gly-77-->Ala, reduces the stability by 0.9 kcal/mol. Simultaneous introduction of these mutations enhances the stability by 0.8 kcal/mol, indicating that the effects of these mutations are cooperative and not simply independent. We determined the crystal structures of these three mutant proteins (G80b-, A77-, and A77/G80b-RNase H) to investigate this cooperative mechanism of the protein stabilization. The structures revealed that the inserted Gly-80b assumes a left-handed helical conformation in both the G80b- and the A77/G80b-RNase H. This inserted glycine residue allows the formation of a "paperclip", which is a common motif at the C-termini of alpha-helices. Accompanying the formation of the paperclip motif, two intrahelical hydrogen bonds are formed between the backbone atoms (O78-N80b and O80b-N84). The stabilization caused by the insertion of Gly-80b can be ascribed to the formation of these hydrogen bonds. The Gly-77-->Ala substitution destabilizes the protein due to the deformed packing interactions in the hydrophobic core around Ala-77 and the stress in the wedged indole ring of Trp-81. These effects are alleviated by the insertion of Gly-80b, which relaxes the backbone structure.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 732, 733, 13371 Extra Details: Escherichia coli ribonuclease HI; cooperative stabilization;,crystal structures; alpha-helices; stability

Submission Details


Submitter: Connie Wang

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

Version: 1

Publication Details
Ishikawa K;Nakamura H;Morikawa K;Kimura S;Kanaya S,Biochemistry (1993) Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution. PMID:8393706
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