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 alpha-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 3J 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 3J alpha beta 2 and 3J 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].