NMR characterization of side chain flexibility and backbone structure in the type I antifreeze protein at near freezing temperatures

The flexibility of the polar side chains in the a-helical Type I antifreeze protein (AFP) near the solution freezing temperature was investigated by two-dimensional nuclear magnetic resonance spectroscopy, These experiments were conducted to define the rotameric conformations of the proposed ice-binding groups, threonines and asparagines, in order to probe the molecular mechanism for ice binding, On the basis of the (3)J(alpha beta)(2) NMR coupling constant values of 7.1, 8.5, 8.5, and 6.8 Hz for residues T2, T13, T24, and T35, respectively, it can be calculated that the regularly spaced ice-binding threonines sample many possible rotameric states prior to ice binding. The lack of a dominant side chain rotamer is further corroborated by nuclear Overhauser distance measurements for T13 and T24. N16 and N27, both with (3)J(alpha beta)(2) and (3)J(alpha beta)(3) coupling constants of 8.4 and 4.5 Hz, respectively, show a slight preference for the side chain conformation with a chi(1) of -60 degrees, These data suggest that prior to ice binding the threonine and asparagine side chains are free to rotate and that a unique preformed ice-binding structure in solution is not apparent. These observations do not support the rigid side chain model proposed recently by an X-ray study [Sicheri, F., & Yang, D. S, C, (1995) Nature 375, 427-431].