IMPORTANCE OF PHOSPHOLIPID IN THE FOLDING AND CONFORMATION OF PHOSPHATIDYLINOSITOL TRANSFER PROTEIN - COMPARISON OF APO AND HOLO SPECIES

The significance of noncovalently bound phospholipid as a structural c omponent of phosphatidyl inositol transfer protein (PITP) and its role in acquisition and maintenance of the native conformation of the prot ein have been addressed by studying the refolding of PITP after exposu re to 6 M guanidinium chloride (GdnCl), Protein conformations were cha racterized by (1)the intrinsic tryptophan fluorescence, circular dichr oism, and absorbance spectroscopy, (2) the degree of binding of the fl uorescent probe 1,8-ANS, and (3) limited proteolytic digestion, When t he GdnCl concentration was reduced 100-fold by rapid dilution at 25 de grees C, practically all of the native transfer activity was regained within 20 min. Endogenous phospholipid demonstrated a strong interacti on with the native PITP. Separation of the phospholipid from the prote in by chromatography on a lipophilic matrix was achieved only under de naturing conditions and resulted in spontaneous oxidation of the ape-p rotein, accompanied by almost complete loss of recoverable transfer ac tivity, Under reducing conditions, however, apo-PITP recovered more th an 80% of the native transfer activity and was similar to holo-PITP in the kinetics of phospholipid transfer. Renatured apo-PITP demonstrate d a significant relaxation of the tertiary structure, compared to nati ve and renatured holo-PITP. Incubation of apo-PITP with phospholipid v esicles resulted in a more compact protein conformation. We conclude t hat the polypeptide can spontaneously fold to a native-like conformati on, sufficient for interaction with a lipid membrane and acquisition o f a phospholipid ligand. Binding of a phospholipid ligand brings about the final adjustments of protein conformation to the more compact nat ive structure.