Backbone and side-chain heteronuclear resonance assignments and hyperfine NMR shifts in horse cytochrome c.
The [H26N, H33N] mutant of horse heart cytochrome c was expressed in E. coli during growth on isotopically enriched minimal media. Complete resonance assignments of both the diamagnetic reduced (spin zero) and paramagnetic oxidized (spin (1/2)) states of the protein were obtained using standard triple resonance and total correlation spectroscopy using the previously determined (1)H chemical shifts of the wild-type protein as a guide. The correspondence of chemical shifts between the wild type and the mutant protein is excellent, indicating that they have nearly identical structures. The expanded library of chemical shifts for both redox states in both proteins allowed the refinement of the electron spin g-tensor of the oxidized states. The g-tensors of the oxidized states of the wild-type and [H26N, H33N] mutant proteins are closely similar, indicating that the subtle details of the ligand fields are nearly identical. The refined g-tensors were then used to probe for redox-dependent structure change in the two proteins.
Liu
Weixia
W
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Rumbley
Jon
J
Englander
S Walter
SW
Wand
A Joshua
AJ
D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006736:Horses; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction
D006418:Heme; D045304:Cytochromes c