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

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 angstroms 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 (phi = 83.1 degrees, psi = -0.3 degrees) close to the left-handed alpha-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 angstroms 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 hydrochloride 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.

Cite this paper

@article{Cooper1996XraySA, title={X-ray structure analysis of an engineered Fe-superoxide dismutase Gly-Ala mutant with significantly reduced stability to denaturant.}, author={Jon B. Cooper and Steve Saward and Peter T. Erskine and Mohammed O. Badasso and Steve P. Wood and Y Zhang and Douglas Brownlie Young}, journal={FEBS letters}, year={1996}, volume={387 2-3}, pages={105-8} }