X-ray structure analysis of an engineered Fe-superoxide dismutase Gly-Ala mutant with significantly reduced stability to denaturant.
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.
Cooper
J B
JB
Department of Crystallography, Birkbeck College, University of London, UK.
Saward
S
S
Erskine
P T
PT
Badasso
M O
MO
Wood
S P
SP
Zhang
Y
Y
Young
D
D
D000409:Alanine / Q000737:chemistry; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial; D004563:Electrochemistry; D005998:Glycine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics
D004269:DNA, Bacterial; D013482:Superoxide Dismutase; D000409:Alanine; D005998:Glycine