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.
Cooper, J. B., Saward, S., Erskine, P. T., Badasso, M. O., Wood, S. P., Zhang, Y., & Young, D. (1996). X-ray structure analysis of an engineered Fe-superoxide dismutase Gly-Ala mutant with significantly reduced stability to denaturant. FEBS Letters, 387(2–3), 105–108. https://doi.org/10.1016/0014-5793(96)00490-5