RESUMEN
A method has been developed allowing structural and binding parameters to be recovered by global analysis of two-dimensional array of steady-state RET data in the special case where energy acceptors distribute between aqueous and lipid phases while donors are embedded in the membrane at a known depth. To test the validity of this approach, correlation and error analyses have been performed using simulated data. To exemplify the method application to the membrane studies, energy transfer from anthrylvinyl-labeled phosphatidylcholine incorporated into mixed phosphatidylcholine/cardiolipin unilamellar vesicles to heme group of cytochrome c is analyzed.
Asunto(s)
Membranas Artificiales , Transferencia de Energía , Reproducibilidad de los ResultadosRESUMEN
Resonance energy transfer between lipid-bound fluorescent probe 3-methoxybenzanthrone as a donor and heme group of cytochrome c as an acceptor has been examined to ascertain the protein disposition relative to the surface of model membranes composed of phosphatidylcholine and cardiolipin (10, 50 and 80 mol%). The model of energy transfer in membrane systems has been extended to the case of donors distributed between the two-bilayer leaflets and acceptors located at the outer monolayer taking into account the donor and acceptor orientational behavior. Assuming specific protein orientation relative to the membrane surface and varying lateral distance of the donor-acceptor closest approach in the range from 0 to 3.5 nm the limits for possible heme distances from the bilayer midplane have been found to be 0.8-3 nm (10 mol% CL), 0-2.6 nm (50 mol% CL), and 1.4-3.3 nm (80 mol% CL).
Asunto(s)
Cardiolipinas/química , Grupo Citocromo c/química , Modelos Biológicos , Fosfatidilcolinas/química , Animales , Benzo(a)Antracenos/química , Cardiolipinas/metabolismo , Bovinos , Grupo Citocromo c/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Hemo/química , Membrana Dobles de Lípidos/química , Membranas Artificiales , Modelos Químicos , Fosfatidilcolinas/metabolismo , Teoría CuánticaRESUMEN
The model of resonance energy transfer (RET) in membrane systems containing donors randomly distributed over two parallel planes separated by fixed distance and acceptors confined to a single plane is presented. Factors determining energy transfer rate are considered with special attention being given to the contribution from orientational heterogeneity of the donor emission and acceptor absorption transition dipoles. Analysis of simulated data suggests that RET in membranes, as compared to intramolecular energy transfer, is substantially less sensitive to the degree of reorientational freedom of chromophores due to averaging over multiple donor-acceptor pairs. The uncertainties in the distance estimation resulting from the unknown mutual orientation of the donor and acceptor are analyzed.
Asunto(s)
Biofisica/métodos , Membrana Dobles de Lípidos/química , Membranas/fisiología , Modelos Biológicos , Simulación por Computador , Transferencia de Energía , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Cinética , Membranas Artificiales , Modelos Estadísticos , Conformación Molecular , Teoría CuánticaRESUMEN
Some applications of resonance energy transfer (RET) method to distance estimation in membrane systems are considered. The model of energy transfer between donors and acceptors randomly distributed over parallel planes localized at the outer and inner membrane leaflets is presented. It is demonstrated that RET method can provide evidence for specific orientation of the fluorophore relative to the lipid-water interface. An approach to estimating the depth of the protein penetration in lipid bilayer is suggested.
