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Local Polarity and Hydrogen Bonding Inside the Sec14p Phospholipid-Binding Cavity: High-Field Multi-Frequency Electron Paramagnetic Resonance Studies

Tatyana I. Smirnova ^*, Thomas G. Chadwick ^*, Maxim A. Voinov ^*, Oleg Poluektov , Johan van Tol , Andrzej Ozarowski , Gabriel Schaaf , Margaret M. Ryan  and Vytas A. Bankaitis 

^* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204; Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439; National High Magnetic Field Laboratory, Tallahassee, Florida 32310; and Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7090

Correspondence: Address reprint requests to Tatyana I. Smirnova, Dept. of Chemistry, North Carolina State University, 2620 Yarbrough Dr., Campus Box 8204, Raleigh, NC 27695-8204. Tel.: 919-513-4375; Fax: 919-513-7353; E-mail: Tatyana_Smirnova{at}ncsu.edu.

Sec14p promotes the energy-independent transfer of either phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between lipid bilayers in vitro and represents the major PtdIns/PtdCho transfer protein in the budding yeast Saccharomyces cerevisiae. Herein, we employ multi-frequency high-field electron paramagnetic resonance (EPR) to analyze the electrostatic and hydrogen-bonding microenvironments for series of doxyl-labeled PtdCho molecules bound by Sec14p in a soluble protein-PtdCho complex. A structurally similar compound, 5-doxyl stearic acid dissolved in a series of solvents, was used for experimental calibration. The experiments yielded two-component rigid limit 130- and 220-GHz EPR spectra with excellent resolution in the g_x region. Those components were assigned to hydrogen-bonded and nonhydrogen-bonded nitroxide species. Partially resolved 130-GHz EPR spectra from n-doxyl-PtdCho bound to Sec14p were analyzed using this two-component model and allowed quantification of two parameters. First, the fraction of hydrogen-bonded nitroxide species for each n-doxyl-PtdCho was calculated. Second, the proticity profile along the phospholipid-binding cavity of Sec14p was characterized. The data suggest the polarity gradient inside the Sec14p cavity is a significant contributor to the driving molecular forces for extracting a phospholipid from the bilayer. Finally, the enhanced g-factor resolution of EPR at 130 and 220 GHz provides researchers with a spectroscopic tool to deconvolute two major contributions to the x-component of the nitroxide g-matrix: hydrogen-bond formation and local electrostatic effects.