Masters Thesis

Determination of the solution structure of antifreeze glycoprotein fraction 8 (afgp8) in deuterated dimethyl sulfoxide (dmso) using nuclear magnetic resonance (nmr) spectroscopy

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)

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