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-dependentosuperoxide dismutase from ~lycobaeterium tuberculosis at 2.9 A 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 (~b = 83.1 °, V = -0.3°) close to the left-handed ~-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 A 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.A,;y words: Superoxide dismutase; ~@cobacterium tuberculosis; X-ray structure; Site-directed m utagenesis