Solution conformation of the Met 61 to His 61 mutant of Pseudomonas stutzeri ZoBell ferrocytochrome c-551.
The gene encoding for bacterial cytochrome c-551 from Pseudomonas stutzeri substrain ZoBell has been mutated to convert the invariant sixth ligand methionine residue into histidine, creating the site-specific mutant M61H. Proton NMR resonance assignments were made for all main-chain and most-side chain protons in the diamagnetic, reduced form at pH 9.2 and 333 K by two-dimensional NMR techniques. Distance constraints (1074) were determined from nuclear Overhauser enhancements and main-chain torsion-angle constraints (72) from scalar coupling estimates. Solution conformations for the protein were computed by the simulated annealing approach. For 28 computed structures, the root mean squared displacement from the average structure excluding the terminal residues 1, 2, 81, and 82 was 0.52 A (sigma = 0.096) for backbone atoms and 0.90 A (sigma = 0.122) for all heavy atoms. The global folding of the mutant protein is the same as for wild type. The biggest changes are localized in a peptide span over residues 60-65. The most striking behavior of the mutant protein is that at room temperature and neutral pH it exists in a state similar to the molten globular state that has been described for several proteins under mild denaturing conditions, but the mutant converts to a more ordered state at high pH and temperature.
Miller
G T
GT
Department of Chemistry, University of Alabama, Tuscaloosa, Alabama 35487, USA.
Hardman
J K
JK
Timkovich
R
R
D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011549:Pseudomonas / Q000737:chemistry* / Q000235:genetics; D012996:Solutions
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D012996:Solutions; D006639:Histidine; C017167:cytochrome C(551); D008715:Methionine