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Investigations into the Membrane Interactions of m-Calpain Domain V

Sarah R. Dennison ^*, Silvia Dante , Thomas Hauß , Klaus Brandenburg , Frederick Harris ^* and David A. Phoenix 

^* Department of Forensic and Investigative Science, University of Central Lancashire, Preston, United Kingdom; Berlin Neutron Scattering Centre, Hahn-Meitner-Institut, Berlin, Germany; Division of Biophysics, Forschunginstitut, Borstel, Germany; and Dean's Office, Faculty of Science, University of Central Lancashire, Preston, United Kingdom

Correspondence: Address reprint requests to D. A. Phoenix, Dean's Office, Faculty of Science, University of Central Lancashire, Preston PR1 2HE, UK. Tel.: 1772-893481; Fax: 1772-894981; E-mail: daphoenix{at}uclan.ac.uk.

m-Calpain is a calcium-dependent heterodimeric protease implicated in a number of pathological conditions. The activation of m-calpain appears to be modulated by membrane interaction, which has been predicted to involve oblique-orientated -helix formation by a GTAMRILGGVI segment located in domain V of the protein's small subunit. Here, we have investigated this prediction. Fourier transform infrared conformational analysis showed that VP1, a peptide homolog of this segment, exhibited -helicity of 45% in the presence of dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine (DMPS) vesicles. The level of helicity was unaffected over a 1- to 8-mM concentration range and did not alter when the anionic lipid composition of these vesicles was varied between 1% and 10% DMPS. Similar levels of -helicity were observed in trifluoroethanol and the peptide appeared to adopt -helical structure at an air/water interface with a molecular area of 164 Ų at the monolayer collapse pressure. VP1 was found to penetrate dimyristoylphosphatidylcholine/DMPS monolayers, and at an initial surface pressure of 30 mN m^–1, the peptide induced surface pressure changes in these monolayers that correlated strongly with their anionic lipid content (maximal at 4 mN m^–1 in the presence of 10% DMPS). Neutron diffraction studies showed VP1 to be localized at the hydrophobic core of model palmitoyloleylphosphatidylcholine/palmitoyloleylphosphatidylserine (10:1 molar ratio) bilayer structures and, in combination, these results are consistent with the oblique membrane penetration predicted for the peptide. It would also appear that although not needed for structural stabilization anionic lipid was required for membrane penetration.