Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies.
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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 gx 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.
author list (cited authors)
Smirnova, T. I., Chadwick, T. G., Voinov, M. A., Poluektov, O., van Tol, J., Ozarowski, A., ... Bankaitis, V. A.
complete list of authors
Smirnova, Tatyana I||Chadwick, Thomas G||Voinov, Maxim A||Poluektov, Oleg||van Tol, Johan||Ozarowski, Andrzej||Schaaf, Gabriel||Ryan, Margaret M||Bankaitis, Vytas A