title: Importance of phospholipid in the folding and conformation of phosphatidylinositol transfer protein: comparison of apo and holo species.
abstract: The significance of noncovalently bound phospholipid as a structural component of phosphatidylinositol transfer protein (PITP) and its role in acquisition and maintenance of the native conformation of the protein have been addressed by studying the refolding of PITP after exposure to 6 M guanidinium chloride (GdnCl). Protein conformations were characterized by (1) the intrinsic tryptophan fluorescence, circular dichroism, and absorbance spectroscopy, (2) the degree of binding of the fluorescent probe 1,8-ANS, and (3) limited proteolytic digestion. When the GdnCl concentration was reduced 100-fold by rapid dilution at 25 degrees C, practically all of the native transfer activity was regained within 20 min. Endogenous phospholipid demonstrated a strong interaction with the native PITP. Separation of the phospholipid from the protein by chromatography on a lipophilic matrix was achieved only under denaturing conditions and resulted in spontaneous oxidation of the apo-protein, accompanied by almost complete loss of recoverable transfer activity. Under reducing conditions, however, apo-PITP recovered more than 80% of the native transfer activity and was similar to holo-PITP in the kinetics of phospholipid transfer. Renatured apo-PITP demonstrated a significant relaxation of the tertiary structure, compared to native and renatured holo-PITP. Incubation of apo-PITP with phospholipid vesicles resulted in a more compact protein conformation. We conclude that the polypeptide can spontaneously fold to a native-like conformation, sufficient for interaction with a lipid membrane and acquisition of a phospholipid ligand. Binding of a phospholipid ligand brings about the final adjustments of protein conformation to the more compact native structure.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Voziyan
authors.forename: J M
authors.initials: JM
authors.lastname: Tremblay
authors.forename: L R
authors.initials: LR
authors.lastname: Yarbrough
authors.forename: G M
authors.initials: GM
authors.lastname: Helmkamp
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines; D007536:Isomerism; D008565:Membrane Proteins; D010084:Oxidation-Reduction; D049294:Phospholipid Transfer Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D002352:Carrier Proteins; D006146:Guanidines; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D019791:Guanidine; D003545:Cysteine