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42141-B7
Investigations of the Binding of Peripheral Membrane Proteins

John J. Breen, Providence College

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 involved fluorescence spectroscopy investigations of the adsorption and desorption of cyt c or [Zn²⁺-heme] cytochrome c from natural and synthetic vesicle systems.  Complementary experiments using surface plasmon resonance (SPR) techniques at the NSF/EPSCoR Proteomics Facility at Brown University are also underway.  We expect the SPR will allow us to more easily extend our work beyond cyt c to other important and more precious peripheral membrane proteins without any labeling requirements on the lipids or proteins.

In the past twelve months we have conducted three sets of experiments, of all which 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.

2)      We have completed a set 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.  These experiments reveal that as the acyl chain length decreases the strong retention of adsorbed cyt c observed for DOPG vesicles is gradually reduced.

3)      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.

In the coming year we will prepare and conduct experiments with a reversibly polymerizable lipid system (S. Regen, et al., JACS (1985) 107, 42-47) following an enzyme catalyzed lipid head group exchange (PC to PG) headgroup exchange (Comfurius and Zwaal, Biochim. Biophys. Acta (1977) 488, 36-42).  In addition we will build on our preliminary experiments with cyt c and using SPR to extend our work to Annexin V, another important and commercially available peripheral membrane protein.

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