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Membrane Position of a Basic Aromatic Peptide that Sequesters Phosphatidylinositol 4,5 Bisphosphate Determined by Site-Directed Spin Labeling and High-Resolution NMR

Jeffrey F. Ellena ^*, Jason Moulthrop ^*, Jing Wu ^*, Michelle Rauch ^*, Sajith Jaysinghne ^*, J. David Castle  and David S. Cafiso ^*

^* Department of Chemistry and Biophysics Program, and Department of Cell Biology, University of Virginia, Charlottesville, Virginia

Correspondence: Address reprint requests to David S. Cafiso, Dept. of Chemistry, University of Virginia, PO Box 400319, Charlottesville, VA 22904-4319. Tel.: 434-924-3067; Fax: 434-924-3567; E-mail: cafiso{at}virginia.edu.

The membrane interactions and position of a positively charged and highly aromatic peptide derived from a secretory carrier membrane protein (SCAMP) are examined using magnetic resonance spectroscopy and several biochemical methods. This peptide (SCAMP-E) is shown to bind to membranes containing phosphatidylinositol 4,5-bisphosphate, PI(4,5)P₂, and sequester PI(4,5)P₂ within the plane of the membrane. Site-directed spin labeling of the SCAMP-E peptide indicates that the position and structure of membrane bound SCAMP-E are not altered by the presence of PI(4,5)P₂, and that the peptide backbone is positioned within the lipid interface below the level of the lipid phosphates. A second approach using high-resolution NMR was used to generate a model for SCAMP-E bound to bicelles. This approach combined oxygen enhancements of nuclear relaxation with a computational method to dock the SCAMP-E peptide at the lipid interface. The model for SCAMP generated by NMR is consistent with the results of site-directed spin labeling and places the peptide backbone in the bilayer interfacial region and the aromatic side chains within the lipid hydrocarbon region. The charged side chains of SCAMP-E lie well within the interface with two arginine residues lying deeper than a plane defined by the position of the lipid phosphates. These data suggest that SCAMP-E interacts with PI(4,5)P₂ through an electrostatic mechanism that does not involve specific lipid-peptide contacts. This interaction may be facilitated by the position of the positively charged side chains on SCAMP-E within a low-dielectric region of the bilayer interface.

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