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Backbone and side–chain heteronuclear resonance assignments and hyperfine NMR shifts in horse cytochrome c
Author(s) -
Liu Weixia,
Rumbley Jon,
Englander S. Walter,
Wand A. Joshua
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.03211303
Subject(s) - heteronuclear molecule , chemistry , chemical shift , crystallography , electron paramagnetic resonance , hyperfine structure , cytochrome c , resonance (particle physics) , mutant , cytochrome , nuclear magnetic resonance spectroscopy , ligand (biochemistry) , diamagnetism , protein structure , stereochemistry , nuclear magnetic resonance , physics , biochemistry , atomic physics , mitochondrion , enzyme , receptor , quantum mechanics , magnetic field , gene
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 ½) 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.