Slow Correlated Movement of Structural Elements in Hemoglobin and Myoglobin

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Structural fluctuations of proteins in solution are often reflections of functional movements and appear to follow low energy pathways that have evolved to accommodate conformational changes triggered, for instance, by binding to ligands or catalysis of reactions. The study of these fluctuations can provide substantial insight into protein function. The spatial extent and dynamics of intramolecular movements in proteins are highly dependent on the environment of the protein, being particularly sensitive to the concentration of proteins and other constituents in the solution. Consequently, studies as a function of protein concentration may provide further insight into the nature of these fluctuations than is possible when experiments are carried out at a single protein concentration. Wide-angle x-ray solution scattering (WAXS) and neutron spin echo spectroscopy (NSE) provide substantial information about the spatial extent and time scale, respectively, of structural fluctuations in solution. They are particularly well suited to the study of slow, correlated movements that are central to many protein functions, providing information complementary to that obtainable by x-ray crystallography and NMR. Using these approaches, we have characterized the spatial and temporal properties of hemoglobin and myoglobin across a wide range of environments. For these proteins, the spatial extent and dynamics of correlated movements both increase rapidly as protein concentration decreases below about 50 mg/ml. A combined analysis of WAXS and NSE data indicates that the slow correlated fluctuations in these molecules are dominated by movement of relatively rigid alpha helices within the subunits. These fluctuations are highly dependent on the ligation state of the molecules and are altered by mutations that impact the function of the molecules.