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Orientation and helical conformation of a tissue-specific hunter-killer peptide in micelles

¹ Department of Chemistry, University of San Diego, San Diego, California 92110, USA
² Genomics Institute of the Novartis Research Foundation, San Diego, California 92121-1125, USA
³ Department of Chemistry, Sonoma State University, Sonoma, California 94928, USA
⁴ Department of Chemistry, McMaster University, Hamilton, Ontario, Canada LS8 4M1
⁵ Department of Chemistry, University of California San Diego (UCSD), San Diego, California 92093, USA

(RECEIVED May 10, 2004; FINAL REVISION May 10, 2004; ACCEPTED May 24, 2004)

Hunter-killer peptides are chimeric synthetic peptides that selectively target specific cell types for an apoptotic death. These peptides, which are models for potential therapeutics, contain a homing sequence for receptor-mediated interactions and a pro-apoptotic sequence. Homing domains have been designed to target angiogenic tumor cells, prostate cells, arthritic tissue and, most recently, adipose tissue. After a receptor-mediated internalization, the apoptotic sequence, which contains D-enantiomer amino acids, initiates apoptosis through mitochondrial membrane disruption. We have begun structure and functional studies on a peptide (HKP1) that specifically targets angiogenic tumor cells for apoptosis. As a model for mitochondrial membrane disruption, we have examined peptide-induced leakage of a calcein fluorophore from large unilamellar vesicles. These experiments demonstrate more potent leakage activity by HKP1 than the peptide lacking the homing domain. Circular dichroism and 2D homonuclear NMR experiments demonstrate that this tumor-specific HKP adopts a left-handed amphipathic helix in association with dodecylphosphorylcholine micelles in a parallel orientation to the lipid–water interface with the homing domain remaining exposed to solvent. The amphipathic helix of the apoptotic domain orients with nonpolar leucine and alanine residues inserting most deeply into the lipid environment.

Keywords: apoptosis; NMR; circular dichroism; peptide

Abbreviations: CD, circular dichroism • CSI, chemical shift indexing • dNN, NOE cross-peak between adjacent amide protons • DPC, dodecylphosphocholine • dDPC, dodecylphosphocholine-d38 • DQF-COSY, double quantum filtered correlated spectroscopy • HKP, hunter-killer peptide • HKP1, hunter-killer peptide with sequence CNGRCGG(KLAKLAK-KLAKLAK)d • LUV, large unilamellar vesicles • NOESY, nuclear Over-hauser effect spectroscopy • PC, phosphatidylcholine • PG, phosphatidyl-glycerol • SDS, sodium dodecyl sulfate • SFM, serum-free medium • TOCSY, total correlation spectroscopy

Reprint request to: Leigh A. Plesniak, Department of Chemistry, University of San Diego, 5998 Alcala Park, San Diego, CA 92110, USA; e-mail: leigh{at}SanDiego.edu; fax: (619) 260-2211.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04853204.

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