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  • Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies.

Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies.

Biophysical journal (2007-02-28)
Tatyana I Smirnova, Thomas G Chadwick, Maxim A Voinov, Oleg Poluektov, Johan van Tol, Andrzej Ozarowski, Gabriel Schaaf, Margaret M Ryan, Vytas A Bankaitis
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Avanti
16:0-7 Doxyl PC, Avanti Research - A Croda Brand 810602P, powder
Avanti
16:0-16 Doxyl PC, Avanti Research - A Croda Brand 810604C
Avanti
16:0-16 Doxyl PC, Avanti Research - A Croda Brand 810604P, powder
Avanti
16:0-10 Doxyl PC, Avanti Research - A Croda Brand 810603P, powder
Avanti
16:0-5 Doxyl PC, Avanti Research - A Croda Brand 810601P, powder
Avanti
16:0-12 Doxyl PC, Avanti Research - A Croda Brand 810600P, powder
Avanti
16:0-5 Doxyl PC, Avanti Research - A Croda Brand 810601C
Avanti
16:0-10 Doxyl PC, Avanti Research - A Croda Brand 810603C