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 met hionine residue into histidine, creating the site-specific mutant M61H, Pro ton NMR resonance assignments were made for all main-chain and most-side ch ain protons in the diamagnetic, reduced form at pH 9.2 and 333 K by two-dim ensional NMR techniques. Distance constraints (1074) were determined from n uclear Overhauser enhancements and main-chain torsion-angle constraints (72 ) from scalar coupling estimates. Solution conformations for the protein we re computed by the simulated annealing approach. For 28 computed structures , the root mean squared displacement from the average structure excluding t he terminal residues 1,2, 81, and 82 was 0.52 Angstrom (sigma = 0.096) for backbone atoms and 0.90 Angstrom (sigma = 0.122) for all heavy atoms. The g lobal folding of the mutant protein is the same as for wild type. The bigge st changes are localized in a peptide span over residues 60-65. The most st riking behavior of the mutant protein is that at room temperature and neutr al pH it exists in a state similar to the molten globular state that has be en described for several proteins under mild denaturing conditions, but the mutant converts to a more ordered state at high pH and temperature.