TRUNCATIONS OF THE C-TERMINUS HAVE DIFFERENT EFFECTS ON THE CONFORMATION AND ACTIVITY OF PHOSPHATIDYLINOSITOL TRANSFER PROTEIN

Contributions of the C-terminus toward the conformation and activity o f phosphatidylinositol transfer protein (PITP) were studied by compari ng properties of the 271 amino acid, full-length protein, PITP(1-271), and two truncated species, PITP(1-259) and PITP(1-253), Using recombi nant proteins and an in vitro phospholipid transfer assay with phospha tidylcholine vesicles, the activities of PITP(1-271) and PITP(1-259) w ere identical, while the activity of PITP(1-253) was almost totally ab olished. By most physical and chemical criteria, however, PITP(1-259) and PITP(1-253) were virtually indistinguishable and differed signific antly from the full-length protein. Results of second derivative analy sis of absorbance spectra were consistent with an additional two Tyr r esidues being exposed to the solvent in PITP(1-259) and PITP(1-253) in comparison to PITP(1-271). Only one out of four Cys residues in PITP( 1-271) reacted with dithiobisnitrobenzoic acid, while two Cys residues were accessible in both truncated species. Quenching of intrinsic Trp fluorescence by acrylamide demonstrated an increase in exposure of Tr p residues in both PITP(1-259) and PITP(1-253); binding of the fluores cence probe 1,8-ANS to these proteins was also significantly higher co mpared to PITP(1-271). These results describe a more relaxed overall t ertiary structure brought about by the C-terminal truncations. This al tered structure did not affect the stability of the truncated proteins , as indicated by equilibrium unfolding in guanidinium chloride. Refol ding of the denatured PITP(1-259), however, was considerably slower th an that of full-length PITP. Our study suggests a critical role of the C-terminal residues 254-259 in transfer activity of PITP. Residues 26 0-271, on the other hand, appear to be more important for the rapid fo lding and maintenance of a compact native conformation of the protein.