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 of phosphatidylinositol transfer protein (PITP) were studied by comparing properties of the 271 amino acid, full-length protein, PITP(1-271), and two truncated species, PITP(1-259) and PITP(1-253). Using recombinant proteins and an in vitro phospholipid transfer assay with phosphatidylcholine vesicles, the activities of PITP(1-271) and PITP(1-259) were identical, while the activity of PITP(1-253) was almost totally abolished. By most physical and chemical criteria, however, PITP(1-259) and PITP(1-253) were virtually indistinguishable and differed significantly from the full-length protein. Results of second derivative analysis of absorbance spectra were consistent with an additional two Tyr residues 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 Trp residues in both PITP(1-259) and PITP(1-253); binding of the fluorescence probe 1,8-ANS to these proteins was also significantly higher compared to PITP(1-271). These results describe a more relaxed overall tertiary structure brought about by the C-terminal truncations. This altered structure did not affect the stability of the truncated proteins, as indicated by equilibrium unfolding in guanidinium chloride. Refolding of the denatured PITP(1-259), however, was considerably slower than that of full-length PITP. Our study suggests a critical role of the C-terminal residues 254-259 in transfer activity of PITP. Residues 260-271, on the other hand, appear to be more important for the rapid folding and maintenance of a compact native conformation of the protein.