Utilizing ESEEM Spectroscopy to Locate the Position of Specific Regions of Membrane Active Peptides within Model Membranes
Raanan Carmieli 1, Niv Papo 2, Herbert Zimmermann 3, Alexey Potapov 2, Yechiel Shai 4 and Daniella Goldfarb 4*
1 Weizmann Institue of Science
2 Weizmann Institute
3 MPI for Medical Research,
4 Weizmann Institute of Science
* To whom correspondence should be addressed. E-mail: daniella.goldfarb{at}weizmann.ac.il.
Submitted on April 15, 2005
Revised on June 6, 2005
Accepted on 5 October 2005
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Abstract |
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Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on Electron Spin-Echo Envelope Modulation (ESEEM) to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration profile of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26-mer bee venom melittin and a de-novo designed 15-mer D,L-amino acid amphipathic peptide (5D-L9K6C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H2O solutions with deuterated model membranes or in D2O solutions with non-deuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG), [DPPC/PG (7:3 w/w)], egg phosphatidylcholine (PC) and PG [PC/PG (7:3 w/w)], and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the 2H nuclei was determined and compared with a series of controls that produced a reference "ruler". Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it "flatter" and increasing the penetration depth into the hydrophobic region.
Key Words:
ESEEM, Peptides, Pulsed EPR, melittin, membranes, spin probes
