N epsilon,N epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation

• Published in: Biochemical journal Vol. 332 ( Pt 2); no. 2; pp. 439 - 449
• Main Authors: Moore, G R, Cox, M C, Crowe, D, Osborne, M J, Rosell, F I, Bujons, J, Barker, P D, Mauk, M R, Mauk, A G
• Format: Journal Article
• Language: English
• Published: England 01.06.1998
• Subjects: Animals; Cattle; Cytochrome c Group - chemistry; Cytochrome-c Peroxidase - chemistry; Cytochromes b5 - chemistry; Cytochromes c; Electron Transport - physiology; Fungal Proteins - chemistry; Horses; Hydrogen-Ion Concentration; Kinetics; Liver - chemistry; Lysine - analogs & derivatives; Lysine - chemistry; Lysine - metabolism; Magnetic Resonance Spectroscopy; Molecular Probes - chemistry; Oxidation-Reduction; Protein Binding; Saccharomyces cerevisiae Proteins
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3320439

The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase. The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein-protein complex formation or rates of interprotein electron transfer. Two-dimensional 1H-13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c. The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes. The NMR characteristics of the Nepsilon,Nepsilon-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nepsilon, Nepsilon-dimethyl-lysine's being a generally useful derivative for characterizing protein-protein complexes.