Role of Electrostatic and Hydrogen Bonding Environment in Sequestering Lipids from Membranes Into the Sec14 Protein Cavity

Abstract

Sec14p is a major yeast phosphatidylinositol/phosophatidylcholine (PtdIns/PtdCho) transfer protein that promotes transfer of PtdIns or PtdCho between lipid bilayers in vitro in energy-independent manner. The exact biophysical mechanism of such a process is unknown at this moment. Here we report on employing an arsenal of advanced spin-labeling EPR methods to probe local electrostatic and hydrogen bonding environment that govern binding of lipids by Sec14 protein. n-doxyl PtdCho (where n=5,7,10,12, and 16 reflects position of nitroxide along the acyl chain) were used as EPR active probes. The local polarity and hydrogen bonding profile inside the lipid binding cavity of Sec14p were assessed from characteristic changes in high field EPR at 130 GHz (D-band). The data indicate that the phospholipid-binding cavity of Sec14p with the likely sequestered water molecules provides a close match for the polarity profile along the bound PtdCho molecule and the one determined for membrane bilayers. This polarity match rationalizes the efficient energy-independent partitioning of a lipid molecule from a bilayer into the Sec14p phospholipid-binding pocket. Further, we have developed a direct method for observing formation of a hydrogen bond between sequestered water molecules and a spin-labeled site by applying pulsed Hyperfine Sub-level Correlation (HYSCORE) spectroscopy. Funded by: NSF-0843632 to TIS and NIH 1R01GM072897 to AIS.