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)
Ishikawa
K
K
Protein Engineering Research Institute, Osaka, Japan.
Nakamura
H
H
Morikawa
K
K
Kimura
S
S
Kanaya
S
S
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005998:Glycine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011487:Protein Conformation; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D016914:Ribonuclease H; C078856:ribonuclease HI; D000409:Alanine; D005998:Glycine