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. Study holds ProTherm entries: 2750, 2751 Extra Details: superoxide dismutase; Mycobacterium tuberculosis;,X-ray structure; site directed mutagenesis
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:19 p.m.
|Number of data points||5|
|Proteins||Superoxide dismutase [Fe]|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MAEYTLPDLDWDYGALEPHISGQINELHHSKHHATYVKGANDAVAKLEEARAKEDHSAILLNEKNLAFNLAGHVNHTIWWKNLSPNGGDKPTGELAAAIADAFGSFDKFRAQFHAAATTVQGSGWAALGWDTLGNKLLIFQVYDHQTNFPLGIVPLLLLDMWEHAFYLQYKNVKVDFAKAFWNVVNWADVQSRYAAATSQTKGLIFG|