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Structural and thermodynamic analyses of Escherichia coli RNase HI variant with quintuple thermostabilizing mutations.

Haruki M, Tanaka M, Motegi T, Tadokoro T, Koga Y, Takano K, Kanaya S
FEBS J 274 5815-25 (2007)
Cited: 8 times
EuropePMC logo PMID: 17944939

Abstract

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.

Articles citing this publication (8)

  1. Thermodynamic system drift in protein evolution. Hart KM, Harms MJ, Schmidt BH, Elya C, Thornton JW, Marqusee S. PLoS Biol 12 e1001994 (2014)
  2. Sequence-based analysis of protein energy landscapes reveals nonuniform thermal adaptation within the proteome. Gu J, Hilser VJ. Mol Biol Evol 26 2217-2227 (2009)
  3. Structure, stability, and folding of ribonuclease H1 from the moderately thermophilic Chlorobium tepidum: comparison with thermophilic and mesophilic homologues. Ratcliff K, Corn J, Marqusee S. Biochemistry 48 5890-5898 (2009)
  4. Improving the thermostability of lipase Lip2 from Yarrowia lipolytica. Wen S, Tan T, Zhao H. J Biotechnol 164 248-253 (2012)
  5. Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins. Okada J, Okamoto T, Mukaiyama A, Tadokoro T, You DJ, Chon H, Koga Y, Takano K, Kanaya S. BMC Evol Biol 10 207 (2010)
  6. The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach. Romero-Romero S, Costas M, Silva Manzano DA, Kordes S, Rojas-Ortega E, Tapia C, Guerra Y, Shanmugaratnam S, Rodríguez-Romero A, Baker D, Höcker B, Fernández-Velasco DA. J Mol Biol 433 167153 (2021)
  7. Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analyses. Rohman MS, Tadokoro T, Angkawidjaja C, Abe Y, Matsumura H, Koga Y, Takano K, Kanaya S. FEBS J 276 603-613 (2009)
  8. Comparisons of Ribonuclease HI Homologs and Mutants Uncover a Multistate Model for Substrate Recognition. Martin JA, Palmer AG. J Am Chem Soc 144 5342-5349 (2022)


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