Stopped-flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions.
A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiation of the reaction by diluting a native protein solution into an exchanging deuterated buffer is described. The method has been used to measure exchange rates of a small set of amide hydrogens of reduced cytochrome c, maintained in a strictly anaerobic atmosphere, in the presence of an otherwise inaccessible range of guanidinium deuterochloride concentrations. The results for the measured protons indicate that hydrogen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the exchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in the 0-2-kcal/mol range.
National Centre for Biological Sciences, TIFR Centre, Indian Institute of Science Campus, Bangalore.
A K
AK
Bhuyan
J B
JB
Udgaonkar
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D003903:Deuterium; D004795:Enzyme Stability; D019791:Guanidine; D006736:Horses; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D011522:Protons; D013816:Thermodynamics
D003574:Cytochrome c Group; D011522:Protons; D006859:Hydrogen; D003903:Deuterium; D019791:Guanidine