X-ray structure analysis of an engineered Fe-superoxide dismutase Gly-Ala mutant with significantly reduced stability to denaturant
| Autor: | S Saward, P. T. Erskine, Ying Zhang, Steve P. Wood, Douglas B. Young, Jonathan B. Cooper, M. Badasso |
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| Rok vydání: | 1996 |
| Předmět: |
DNA
Bacterial Steric effects Protein Denaturation Protein Conformation Stereochemistry Molecular Sequence Data Mutant Glycine Biophysics Crystallography X-Ray Biochemistry chemistry.chemical_compound Tetramer Structural Biology Electrochemistry Genetics Denaturation (biochemistry) Asparagine Guanidine Molecular Biology chemistry.chemical_classification Alanine Site-directed mutagenesis Base Sequence Superoxide Dismutase Mycobacterium tuberculosis Cell Biology Amino acid Crystallography Spectrometry Fluorescence chemistry Mutagenesis Site-Directed X-ray structure |
| Zdroj: | FEBS Letters. 387:105-108 |
| ISSN: | 0014-5793 |
| DOI: | 10.1016/0014-5793(96)00490-5 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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