Structure of type I antifreeze protein and mutants in supercooled water

Many organisms are able to survive subzero temperatures at which bodily flu ids would normally be expected to freeze. These organisms have adapted to t hese lower temperatures by synthesizing antifreeze proteins (AFPs), capable of binding to ice, which make further growth of ice energetically unfavora ble. To date, the structures of five AFPs have been determined, and they sh ow considerable sequence and structural diversity. The type I AFP reveals a single 37-residue a-helical structure. We have studied the behavior of wil d-type type I AFP and two "inactive" mutants (Ala17Leu and Thr13Ser/Thr24Se r) in normal and supercooled solutions of H2O and deuterium oxide (D2O) to see if the structure at temperatures below the equilibrium freezing point i s different from the structure observed at above freezing temperatures. Ana lysis of 1 D H-1- and C-13-NMR spectra illustrate that all three proteins r emain folded as the temperature is lowered and even seem to become more alp ha -helical as evidenced by C-13(alpha)-NMR chemical shift changes. Further more, C-13-T-2 NMR relaxation measurements demonstrate that the rotational correlation times of all three proteins behave in a predictable manner unde r all temperatures and conditions studied. These data have important implic ations for the structure of the AFP bound to ice as well as the mechanisms for ice-binding and protein oligomerization.