Functional protein dynamics from Site Directed Spin Labeling

By virtue of their small size and stabilization by weak interactions, protein molecules are inherently compressible and undergo substantial structural fluctuations. Evidence accumulated over the last decade shows that fluctuations on the ps to ms and longer time scales are functional, and determine in part the kinetics, specificity and affinity of ligand binding and protein-protein interactions, and provide a mechanism for allosteric coupling. To understand the molecular basis of protein function is then essential to have experimental tools available to detect motions on the above mentioned time scale. NMR relaxation methods have provided the most detailed description of dynamics for small proteins in solution, but face significant challenges in the study of more complex systems, i.e. transmembrane helical receptors in their native environment. Site Directed Spin Labeling (SDSL) and new strategies in EPR spectroscopy provide an alternative approach, not limited by the size and complexity of the system, with significant advantages of sensitivity and time scale. Earlier studies documented the ability of SDSL to map fast {ps-ns} backbone dynamics. Recent studies suggest that {µs-ms} conformational fluctuations can be detected in spin-labeled proteins through the use of osmotic and hydrostatic pressure perturbation, and exchange rates can be determined using pulse saturation recovery EPR spectroscopy