ESR and Fluorescence Studies of Thrombin Active Site Conformation

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The chapters in this book have introduced thrombin from a detailed primary structure viewpoint, from the three-dimensional x-ray structure, and from NMR studies of ligands, such as hirudin and fibrinopeptide analogs, which complex with this enzyme. It should be noted that the highly impressive accomplishments of the three-dimensional structure determination of PPACK-human α-thrombin and its complexes with hirudin and hirudin analogs are quite recent reports, within one year of the publication of this tome. Prior to that time we had available several spectroscopic techniques allowing us to probe aspects of protein structure and conformation in solution. While most of the spectroscopic techniques cannot distinguish individual amino acid residues and their precise conformation (except high-resolution proton NMR), they are, nonetheless, sensitive methods for examining aspects of conformation and dynamics. That is, the physical bases, which contribute to the spectral output from techniques such as electron spin resonance (ESR), fluorescence, circular dichroism (CD), and other optical spectroscopies, are highly sensitive to small changes or movements within a protein structure. This chapter addresses two biophysical techniques, ESR (spin-labeling) and fluorescence spectroscopy, with examples demonstrating their power and simplicity in unraveling conformational states and binding modes in various thrombin species.