title: CW dipolar broadening EPR spectroscopy and mechanically aligned bilayers used to measure distance and relative orientation between two TOAC spin labels on an antimicrobial peptide.
abstract: An EPR membrane alignment technique was applied to measure distance and relative orientations between two spin labels on a protein oriented along the surface of the membrane. Previously we demonstrated an EPR membrane alignment technique for measuring distances and relative orientations between two spin labels using a dual TOAC-labeled integral transmembrane peptide (M2δ segment of Acetylcholine receptor) as a test system. In this study we further utilized this technique and successfully measured the distance and relative orientations between two spin labels on a membrane peripheral peptide (antimicrobial peptide magainin-2). The TOAC-labeled magainin-2 peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 15.3Å from a dual TOAC-labeled magainin-2 peptide at positions 8 and 14 that closely matches with the 13.3Å distance obtained from a model of the labeled magainin peptide. In addition, the angles determining the relative orientations of the two nitroxides have been determined, and the results compare favorably with molecular modeling. This study demonstrates the utility of the technique for proteins oriented along the surface of the membrane in addition to the previous results for proteins situated within the membrane bilayer.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States.
authors.forename: Indra D
authors.initials: ID
authors.lastname: Sahu
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, United States.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Hustedt
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States.
authors.forename: Harishchandra
authors.initials: H
authors.lastname: Ghimire
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States.
authors.forename: Johnson J
authors.initials: JJ
authors.lastname: Inbaraj
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States.
authors.forename: Robert M
authors.initials: RM
authors.lastname: McCarrick
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States. Electronic address: gary.lorigan@miamioh.edu.
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
keywords: Aligned DMPC bilayers; Antimicrobial peptide; Dipolar broadening CW-EPR; Distance measurement; Magainin-2