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J. Biol. Chem., Vol. 279, Issue 44, 45815-45823, October 29, 2004

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Structure and Orientation of Pardaxin Determined by NMR Experiments in Model Membranes^*

Fernando Porcelli, Bethany Buck, Dong-Kuk Lee¶||, Kevin J. Hallock¶, Ayyalusamy Ramamoorthy¶||**, and Gianluigi Veglia

From the Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, Department of Environmental Sciences (Di.S.A.) and INFM Sez. B University of Tuscia, Viterbo, Italy, and ^¶Department of Chemistry and ^||Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109-1055

Pardaxins are a class of ichthyotoxic peptides isolated from fish mucous glands. Pardaxins physically interact with cell membranes by forming pores or voltage-gated ion channels that disrupt cellular functions. Here we report the high-resolution structure of synthetic pardaxin Pa4 in sodium dodecylphosphocholine micelles, as determined by ¹H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 ± 9°, making this structure substantially different from the one previously determined in organic solvents. In addition, paramagnetic solution NMR experiments on Pa4 in micelles reveal that except for the C terminus, the peptide is not solvent-exposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, ¹³C-¹⁵N rotational echo double-resonance experiments in multilamellar vesicles support the helical conformation of the C-terminal segment, whereas ²H NMR experiments show that the peptide induces considerable disorder in both the head-groups and the hydrophobic core of the bilayers. These solid-state NMR studies indicate that the C-terminal helix has a transmembrane orientation in DMPC bilayers, whereas in POPC bilayers, this domain is heterogeneously oriented on the lipid surface and undergoes slow motion on the NMR time scale. These new data help explain how the non-covalent interactions of Pa4 with lipid membranes induce a stable secondary structure and provide an atomic view of the membrane insertion process of Pa4.

Received for publication, May 17, 2004 , and in revised form, July 17, 2004.

^* This research was partly supported by an National Science Foundation Grant (CAREER development award to A. R) and funds from the National Institutes of Health (Grant AI054515-01A1). NMR instrumentation was provided with funds from the National Science Foundation (Grant BIR-961477) and the University of Minnesota Medical School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table.

The atomic coordinates and structure factors (code 1XC0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^** To whom correspondence may be addressed: Biophysics Research Division and Dept. of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055. Tel.: 734-647-6572, Fax: 734-615-3790; E-mail: ramamoor{at}umich.edu. To whom correspondence may be addressed: Dept. of Chemistry, University of Minnesota, 139 Smith Hall, 207 Pleasant St. S.E., Minneapolis, MN 55455. Tel.: 612-625-0758; Fax 612-626-7541; E-mail: veglia{at}chem.umn.edu.

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