ACS PRF | ACS
All e-Annual Reports
42141-B7
Investigations of the Binding of Peripheral Membrane Proteins
The aim of our project is to experimentally test the hypothesis that phospholipids can and do adopt an extended conformation during the important dynamic processes of binding peripheral proteins to membranes. In the extended conformation the non-polar tails of a lipid contained in a lamellar phase extend in opposite directions. While this conformation is highly disfavored in a normal bilayer environment, P. K. J. Kinnunen and coworkers have postulated the extended conformation is a likely way to relieve the packing strain experienced by lipids with the propensity to form HII- phases (Chemistry and Physics of Lipids 1992, 63, 251-258). More importantly, they propose the extended conformation is 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.
Following the large body of work published by the Kinnunen group our work is also focused on the interactions between cytochrome c (cyt c) and model membrane systems composed of acidic phospholipids. Our experimental approach for this project was divided along two tracts; (1) AFM imaging and force measurement studies of solid supported membranes with and without cyt c and (2) fluorescence spectroscopy investigations of the adsorption and desorption of cyt c or [Zn2+-heme] cytochrome c from natural and synthetic vesicle systems. In the next few weeks we will explore a third tract as we begin a series of surface plasmon resonance (SPR) experiments at the NSF/EPSCoR Proteomics Facility at Brown University. 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.
The bulk of our experimental effort during the 2006-2007 funding period was directed towards our fluorescence spectroscopy experiments. During the academic year, two undergraduate students were successful in completing the following activities or experiments towards out project goal:
1) As set of cyt c competitive binding experiments between pyrene labeled DOPG vesicles containing a substantial fraction of N-acylated glycerol lipids with C2, or C16 acyl chains (fully saturated) and DOPG vesicles.
2) A set of cyt c competitive binding experiments between pyrene labeled DOPG vesicles containing 10 to 40 mole% cholesterol and DOPG vesicles.
3) The preparation of brominated DOPG.
4) The preparation of [Zn2+-heme] cyt c.
5) The preparation of two brominated N-acylated lipids (-COCH2CH2Br and -CO(CH2)11CH2Br).
Our most significant finding to date comes from the cyt c competitive binding experiments between pyrene labeled DOPG vesicles containing 10 to 40 mole% cholesterol and DOPG vesicles. The addition of increasing amounts of cholesterol to the DOPG vesicles increases the extent to which adsorbed cyt c desorbs upon the addition of an excess of unlabeled DOPG vesicles. We attribute this behavior to the decrease in the fluidity and increase in the order of lipids in the membrane upon the incorporation of cholesterol and the resulting restriction in a lipid's ability to adopt the extended conformation.
In the coming months we will confirm and extend as needed our experiments with cholesterol containing vesicles as we prepare to report out findings. We also anticipate beginning a series of fluorescence quenching experiments between [Zn2+-heme] cyt c and our array of brominated lipids, establishing SPR as a useful experimental technique for our project, and the resumption of our AFM experiments.
