Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies.
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.
Smirnova
Tatyana I
TI
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA. Tatyana_Smirnova@ncsu.edu
Chadwick
Thomas G
TG
Voinov
Maxim A
MA
Poluektov
Oleg
O
van Tol
Johan
J
Ozarowski
Andrzej
A
Schaaf
Gabriel
G
Ryan
Margaret M
MM
Bankaitis
Vytas A
VA
D001665:Binding Sites; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006860:Hydrogen Bonding; D008956:Models, Chemical; D008958:Models, Molecular; D049294:Phospholipid Transfer Proteins / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry*; D055672:Static Electricity
D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; C483438:SEC14 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents