The effect of co-solvents on the occupancy of non-coordinated internal water in the distal pocket of myoglobin

Water molecules and their hydrogen bonding potentials play important roles in dynamic and thermodynamic processes of proteins.  Traditional structural techniques are limited in determining the stability and rigidity of proteins, shifting the pK_a values of buried ionizable residues, and modulating dynamical processes such as folding, catalysis, and proton transfers and so the development of new methods should be explored.  Our laboratory has developed a kinetic spectral marker for the entry of non-coordinated water into the distal heme pocket of myoglobin; there is a blue shift in the 560-nm deoxy absorption peak occurring on a nanosecond time-scale after CO photolysis.  To monitor the effects of different co-solvents, we employ time-resolved absorption spectroscopy to measure the occupancy of the non-coordinated water molecule in the distal pocket as a function of co-solvent (glycerol, PEG, albumin, PVD, Ficoll, and Trehalose).  Our results will allow us to determine how physical properties of solvent/co-solvents, such as viscosity, water activity, and density, influence water occupancy within myoglobin’s distal pocket.  Accurately measuring water occupancy in myoglobin as a function of co-solvents will provide insights into how proteins may function differently in vitro compared to the cellular milieu (the molecular crowding effect) due to changes in their internal water occupancy.