Determination of the Solution Structure of Antifreeze Glycoprotein Fraction 8 (AFGP8) in Deuterated Dimethyl Sulfoxide (DMSO) Using Nuclear Magnetic Resonance (NMR) Spectroscopy.

摘要

Fish native to the polar regions produce biological antifreezes called antifreeze glycoproteins (AFGPs) to prevent themselves from freezing at supercool temperatures (--1.9 °C). AFGP lowers the freezing point of water in a non-colligative manner. AFGP's ability to induce thermal hysteresis (change in freezing point with little to no effect on the melting point) makes them about 500 times more effective than traditional colligative antifreeze, such as salts and sugars. By understanding the antifreeze mechanism of AFGP, it is possible to model molecules that can mimic AFGP. The structure-function paradigm states that the function of a protein can be related to its structure. The primary sequence of AFGP is highly degenerate consisting of multiple repeats of the same tripeptide Ala-Ala-Thr*, in which Thr* is glycosylated with the disaccharide beta-D-galactosyl-(1,3)-alpha-N-acetyl-D-galactosamine . The smallest number of tripeptide repeats called AFGP fraction 8 (AFGP8) was studied. In addition to its highly degenerate primary sequence, AFGP seems to function as an intrinsically disordered protein which presents challenges in determining their native structure. In this thesis work, a different approach was used to elucidate the three-dimensional structure of AFGP8 from Arctic cod Boreogadus saida and Antarctic notothenioid Trematomus borchgrevinki. Dimethyl sulfoxide, a non-native solvent, was used to make AFGP8 less dynamic in solution and induced a non-native structure which was determined via nuclear magnetic resonance (NMR) spectroscopy. The overall three-dimensional structure between the two AFGP8, from two different natural sources, was different, but their "compactness" were very similar due to the same diffusion coefficient measured with NMR spectroscopy. In addition to the similar compactness, the conserved motifs, Ala-Thr*-Pro-Ala and Ala-Thr*-Ala-Ala, presented in both AFGP8 seemed to have very similar three-dimensional structure. The determination of the three-dimensional structure of AFGP8 in dimethyl sulfoxide sets a foundation for a future experiment in performing a solvent titration scheme. The solvent titration scheme will allow for tracking structural changes of the non-native structure of AFGP (in DMSO) to the native, disordered structure of AFGP (in water) in hope that the perturbation of the structure will provide insight on the antifreeze mechanism of an antifreeze glycoprotein.