X-ray structure analysis of an engineered Fe-superoxide dismutase Gly-Ala mutant with significantly reduced stability to denaturant

  • Cooper J
  • Saward S
  • Erskine P
 et al. 
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Abstract

We have refined the X-ray structure of a site-directed G152A mutant of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.9 Å resolution. The mutation which replaces a glycine residue in a surface loop with alanine was designed to alter the conformation of this loop region which has previously been shown to play a crucial structural role in quaternary interactions within the SOD tetramer, Gly-152 was targeted as it has dihedral angles (φ = 83.1°, ψ = -0.3°) close to the left-handed α-helical conformation which is rarely adopted by other amino acids except asparagine, Gly-152 was replaced by alanine as it has similar size and polarity, yet has a very low tendency to adopt similar conformations, X-ray data collection on crystals of this mutant at 2.9 Å resolution and subsequent least-squares refinement to an R-value of 0.169 clearly establish that the loop conformation is unaffected. Fluorescence studies of guanidine hydrachloride denaturation establish that the mutant is 4 kcal/mol less stable than the wild-type enzyme. Our results indicate that strict conformational constraints imposed upon a region of polypeptide, due for example to interactions with a neighbouring subunit, may force an alanine residue to adopt this sterically hindered conformation with a consequent reduction in stability of the folded conformation.

Author-supplied keywords

  • Mycobacterium tuberculosis
  • Site-directed mutagenesis
  • Superoxide dismutase
  • X-ray structure

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Authors

  • J. B. Cooper

  • S. Saward

  • P. T. Erskine

  • M. O. Badasso

  • S. P. Wood

  • Y. Zhang

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