2YV0

Structural and Thermodynamic Analyses of E. coli ribonuclease HI Variant with Quintuple Thermostabilizing Mutations


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Work: 0.182 
  • R-Value Observed: 0.182 

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This is version 1.3 of the entry. See complete history


Literature

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.

(2007) FEBS J 274: 5815-5825

  • DOI: https://doi.org/10.1111/j.1742-4658.2007.06104.x
  • Primary Citation of Related Structures:  
    2YV0

  • PubMed 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.


  • Organizational Affiliation

    Department of Materials Chemistry and Engineering, Nihon University, Koriyama, Japan. haruki@chem.ce.nihon-u.ac.jp


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Ribonuclease HIA [auth X]155Escherichia coliMutation(s): 5 
Gene Names: rnhA
EC: 3.1.26.4
UniProt
Find proteins for P0A7Y4 (Escherichia coli (strain K12))
Explore P0A7Y4 
Go to UniProtKB:  P0A7Y4
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0A7Y4
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.40 Å
  • R-Value Work: 0.182 
  • R-Value Observed: 0.182 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 43.371α = 90
b = 86.483β = 90
c = 34.816γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
MOLREPphasing

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2008-03-18
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2021-11-10
    Changes: Database references
  • Version 1.3: 2023-10-25
    Changes: Data collection, Refinement description