Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance 13C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the 1H-13C NOE were determined in this study. The CαH relaxation measurements were compared to the previously measured 15N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the χ1 dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159–7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than ±25°.