The program concerns the relationship of structure to function of proteins (antifreeze proteins) that inhibit ice crystal development. The main studies will be with four closely related antifreeze glycoproteins from the blood serums of four different fishes, Pagothenia borchgrevinki (from Antarctica), Dissostichus mawsoni (from Antarctica), Boreogadus saida (from Arctic), and Eleginus gracilis (from Arctic and the sea off northern Japan). Chemical derivatives and syntheses (e.g., polymers) of these antifreeze proteins, as well as of other proteins, such as gelatin and salivary proteins, will also be used. Solution properties will be examined by conventional methods for macromolecules, including proton, 13C and 15N (with 15N derivatives) NMR. Studies of mechanism of action will include determinations of effects in inhibition of the growth of ice crystals once formed (""""""""antifreeze"""""""" activity-the noncolligative lowering of the freezing temperature without effecting the melting temperature) and of effects on homogeneous and heterogeneous nucleation (""""""""supercooling"""""""" activity). Attention will also be given to possible relationships between antifreeze activity and supercooling activity. Particular attention will be focused on the interaction of the antifreeze proteins at the ice-solution interface, as a possible mechanism for antifreeze action: an inhibition of ice crystal growth by interacting at the ice crystal surface. These latter studies will include: (a) direct demonstration of the proteins on the ice surface by generation of the surface second harmonics using high energy pulse laser, (b) microscopic determination of effects on changes in curvatures (cusps) at grain boundaries at the interfacial areas between two adjacent growing ice crystallites and the solution, (c) attempts to show interactions (cooperative potentiations) between differently acting antifreeze proteins at the ice solution interface using fluorescence energy transfer, and (d) calculations of data on inhibition of ice crystal growth at different concentrations of different antifreeze proteins at different temperatures. Associated studies will be directed at understanding how different antifreeze proteins function differently (i.e., have different quantitative activities) in inhibiting ice crystals with different extents (and amounts) of roughness of the ice surfaces. Related to many of the above will be observations of the effects of the different proteins on recrystallizations (usually the development of large crystals at the expense of small crystals) in aqueous solutions and biological fluids.
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