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PDBsum entry 2yv0
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References listed in PDB file
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Key reference
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Title
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Structural and thermodynamic analyses of escherichia coli rnase hi variant with quintuple thermostabilizing mutations.
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Authors
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M.Haruki,
M.Tanaka,
T.Motegi,
T.Tadokoro,
Y.Koga,
K.Takano,
S.Kanaya.
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Ref.
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Febs J, 2007,
274,
5815-5825.
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PubMed id
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Abstract
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A combination of five thermostabilizing mutations, Gly23-->Ala,
His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His, has been
shown to additively enhance the thermostability of Escherichia coli RNase HI
[Akasako A, Haruki M, Oobatake M & Kanaya S (1995) Biochemistry34,
8115-8122]. In this study, we determined the crystal structure of the protein
with these mutations (5H-RNase HI) to analyze the effects of the mutations on
the structure in detail. The structures of the mutation sites were almost
identical to those of the mutant proteins to which the mutations were
individually introduced, except for G23A, for which the structure of the single
mutant protein is not available. Moreover, only slight changes in the backbone
conformation of the protein were observed, and the interactions of the side
chains were almost conserved. These results indicate that these mutations almost
independently affect the protein structure, and are consistent with the fact
that the thermostabiling effects of the mutations are cumulative. We also
determined the protein stability curve describing the temperature dependence of
the free energy of unfolding of 5H-RNase HI to elucidate the thermostabilization
mechanism. The maximal stability for 5H-RNase HI was as high as that for the
cysteine-free variant of Thermus thermophilus RNase HI. In contrast, the heat
capacity of unfolding for 5H-RNase H was similar to that for E. coli RNase HI,
which is considerably higher than that for T. thermophilus RNase HI. These
results suggest that 5H-RNase HI is stabilized, in part, by the
thermostabilization mechanism adopted by T. thermophilus RNase HI.
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