High-field EPR spectroscopy on transfer proteins in biological action

In the last decade joint efforts of biologists, chemists, and physicists were made to understand the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. Characteristic examples of such factors are time varying specific H-bonding patterns and/or polarity effects of the microenvironment. In this overview, a few large paradigm biosystems are surveyed which have been explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three transfer proteins in action are characterized with respect to structure and dynamics: (1) light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from photosynthetic bacteria Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales.