A series of physical studies will be performed to examine how the carbohydrate side chains of a glycoprotein (and their chain length) affect the secondary and tertiary structure of the glycoprotein, the thermodynamics of unfolding and 3- dimensional structure. To accomplish these goals: (1) a series of derivatives of beta2 glycoproteinI (B2GPI) will be prepared and isolated using specific enzymes to sequentially remove individual carbohydrate residues from each of the N-linked chains; (2) the solution conformation of each derivative (both secondary and tertiary structure) will be examined using far and near UV CD spectroscopy. These studies will be performed as a function to temperature in aqueous buffers. The solvent environment will also be varied to investigate the effect of different solvents (aqueous buffers, ionic strength, pH, organic solvents) have on the conformation of each derivative; (3) the thermodynamics of unfolding of each derivative will be examined using high resolution calorimetry. This will provide energetic information on the effect of carbohydrate chain length on the thermal stability of each derivative. Aqueous buffer, ionic strength and pH will be varied. The thermodynamic data will be correlated with the structural data provided in goal (2); (4) native B2GPI, one partially deglycosylated and the totally deglycosylated derivatives will be crystallized. The effect of carbohydrate chain alteration on the conditions for crystal growth and crystal structure which may arise from alteration of the conformation of the folded polypeptide as a result of removal of defined carbohydrate residues will be examined.
