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The aim of our project is to experimentally test the hypothesis that phospholipids can and do adopt an extended conformation during the important dynamic process of binding peripheral proteins to membranes.  In the extended conformation the non-polar acyl chains of a lipid contained in a lamellar phase extend in opposite directions.  As postulated by P. K. J. Kinnunen and coworkers, the extended conformation is a way to relieve the packing strain experienced by lipids with the propensity to form H_II- phases (Chem. Phys. Lipids (1992) 63, 251-258).  Kinnunen's group also proposes the extended conformation is more likely to occur when there is no hydrophilic barrier to the extended conformation, as would be the case with the adsorption of a protein with a hydrophobic channel.  Recently the extended conformation has been proposed to explain the   targeting of Gag proteins with rafts in the plasma membrane subsequently leading to the assembly of the type 1 HIV. (Saad et al., Proc. Natl. Acad. Sci. USA, 2006, 103 11364-11369)

Following the large body of work published by the Kinnunen group our work is primarily focused on the interactions between cytochrome c (cyt c) and model membrane systems composed of acidic phospholipids.  The majority of our work has relied on fluorescence spectroscopy to investigate the adsorption and desorption of cyt c or [Zn²⁺-heme] cytochrome c from natural and synthetic vesicle systems.  Complementary docking calculations between cyt c and a series of simple fatty acid have also been completed. 

During the four years of support we have received from the ACS-PRF (includes a 1 year no-cost extension) we have conducted three types of experiments and begun a fourth type of experiment.  All our results support the occurrence of the extended lipid conformation.

1)      We have completed a set of cyt c competitive binding experiments between pyrene labeled vesicles containing 50 mole% of N-acylated PE lipids with third C₂, or C₁₆ acyl chains (fully saturated) and unlabeled DOPG vesicles. These experiments reveal the strong retention of adsorbed cyt c observed for DOPG vesicles is absent when the acidic lipids possess a third acyl chain.

1)      We have completed two sets of cyt c competitive binding experiments between pyrene labeled PG vesicles composed of lipids with differing acyl chain lengths (C₁₈, C₁₆, C₁₄, C₁₂, C₁₀, and C₈) and DOPG vesicles.  In one set the vesicles are composed only of the PG lipids and in the other set a 50:50 mix with DOPC.  Both sets of experiments reveal that as the acyl chain length decreases the strong retention of adsorbed cyt c observed for DOPG vesicles is gradually reduced.  Calculations conducted with AutoDock to determine the interaction energy between simple fatty acids with the same acyl chains and a hydrophobic groove on the exterior of cyt c support this observation.

2)      We have completed a set of quenching experiments between the luminescent [Zn²⁺-heme] cyt c and vesicles containing either N-acylated PE lipids with a terminal Br on a short, C₃, acyl chain or the fully brominated product of Br₂ and DOPG, D(Br₂)SPG.  These experiments reveal that quenching of the [Zn²⁺-heme] cyt c luminescence occurs only when Br is positioned on one of the acyl chains capable of adopting the extended conformation and not when constrained to the interfacial region of a membrane.

3)      We have obtained preliminary data from competitive binding experiments using vesicles containing photopolymerizable lipids (O'Brien et al., Biochim. Biophys. Acta (1982) 693 437-443.  These lipids require an exchange of the head group (PC to PG) which is accomplished using phospholipase D (Comfurius and Zwaal, Biochim. Biophys. Acta (1977) 488, 36-42).   Our early experiments reveal a measurable difference in the retention of cyt c by vesicles containing these lipids before and after exposure to UV light.  We are currently repeating these experiments and conducting other experiments to access the effects vesicle composition and UV exposure time. 

The support we have received from the ACS-PRF is greatly appreciated.  A total of seven undergraduate students have worked on this project.  Two of these students are currently in graduate chemistry programs and three more students will begin there graduate studies in chemistry, biochemistry, or neuroscience in the coming year. The project has helped the PI broaden his experience, continue a collaboration with a chemist at a major research university, and forge a successful collaboration with a department faculty colleague.  Poster presentations have been made a three regional undergraduate science conferences and a poster presentation will be made at the Spring 2010 ACS National Meeting.  We also look forward to publicly acknowledging our thanks to the donors of the ACS-PRF in three publications will come from this work.