Synthesis, purification and characterization of the second transmembrane domain of Crep-1, Tm-B and effects of polyunsaturated phospholipids (plipc) and cholesterol on the alignment temperature and fluidity of magnetically aligned DMPC/DHPC phospholipid bilayers

by Adhikari, Prem R.

SYNTHESIS, PURIFICATION AND CHARACTERIZATION OF THE SECOND TRANSMEMBRANE DOMAIN OF CREP-1, TM-B AND EFFECTS OF POLYUNSATURATED PHOSPHOLIPIDS (PLIPC) AND CHOLESTEROL ON THE ALIGNMENT TEMPERATURE AND FLUIDITY OF MAGNETICALLY ALIGNED DMPC/DHPC PHOSPHOLIPID BILAYERS by Prem R. Adhikari CREP-1 is a microsomal ?12-desaturase and acetylenase that has four transmembrane segments. The second transmembrane domain (TM-B) of CREP-1 was synthesized, purified, and its secondary structure was determined using CD spectroscopy. The low ?-helicity of TM-B in various solvents encouraged us to explore a more biomimetic model membrane system, which includes unsaturated phospholipids PLiPC and cholesterol. The presence of unsaturated phospholipids and cholesterol in DMPC/DHPC bicelles increased the fluidity and rigidity of the system, respectively. The temperature study of modified DMPC/DHPC bicelles by 2H solid-state NMR spectroscopy showed a decrease in the alignment temperature and an increase in the fluidity of the bicelle system with PLiPC whereas the cholesterol had the opposite effect. Synthesis, Purification and Characterization of the Second Transmembrane Domain of CREP-1, TM-B, and Effects of Polyunsaturated Phospholipids (PLiPC) and Cholesterol on the Alignment Temperature and Fluidity of Magnetically Aligned DMPC/DHPC Phospholipid bilayers A Thesis Submitted to the Faculty of Miami University In partial fulfillment of the requirement for the degree of Master of Science Department of Chemistry and Biochemistry By Prem R. Adhikari, Miami University Oxford, Ohio 2003 Advisor______________________________________ Robert E. Minto Advisor______________________________________ Gary A. Lorigan Reader _______________________________________ Richard T. Taylor Reader _______________________________________ Michael W. Crowder