Development and application of high-field, high-gradient pulsed field gradient NMR for studies of diffusion behavior of lipids in model membranes .

摘要

This work presents the development and application of a high-field, high-gradient pulsed field gradient nuclear magnetic resonance technique for studies of lipid lateral diffusion in planar-supported lipid bilayers composed of ternary mixtures of lipids intended to mimic the composition of eukaryotic cell membranes. Lipid rafts are small functional domains that exist in cell membranes. It is widely accepted that rafts participate in many cellular activities such as signal transduction. Lipid rafts in non-activated cells are believed to be smaller than around 200 nm in size and quite unstable. Liquid-ordered domains in model membranes share similar characteristics to lipid rafts. In these studies, domains as large as 10 mum have been observed under certain conditions. However, recent data indicates that much smaller domains can also form and remain stable in these model membranes under certain conditions. In order to accurately characterize lateral transport of lipids in domain-forming model membranes and possibly extract information relevant to the study of lipid rafts, an experimental technique is required which has sufficient spatial resolution and does not disturb the membrane or any liquid-ordered domains which might exist. Pulsed field gradient nuclear magnetic resonance (PFG NMR) allows for the direct observation of molecular mean square displacements and their related diffusion coefficients in a manner which does not perturb the membrane since additives, such as fluorescently-labeled lipids, are unnecessary. In this work, the use of a high magnetic field strength (17.6 T) coupled with high magnetic field gradient strength of up to 30 T/m affords the use of smaller diffusion times under the conditions of the narrow-pulse approximation which allows for distortion-free monitoring of time-dependent and displacement-dependent diffusion behavior of lipids with superior signal-to-noise. Diffusion measurements conducted on membranes consisting of a mixture of DOPC, SM, and Chol show diffusion behavior which was independent of diffusion time, consistent with the presence of large liquid-ordered domains. Formation of smaller domains was observed in membranes consisting of DOPC, DPPC, and Chol near the miscibility transition temperature which manifests as time-dependent diffusion behavior of lipids. It was verified that this behavior was a consequence of lipid exchange between liquid-ordered domains and the surrounding liquid-disordered environment rather than an NMR relaxation effect. Dynamic Monte Carlo simulations were used in conjunction with time-dependent diffusion results to extract information about domain size and the permeability of the domain boundary. This is the first observation of time-dependent diffusion and estimation of such properties for lipid membranes of any composition. (Full text of this dissertation may be available via the University of Florida Libraries web site. Please check http://www.uflib.ufl.edu/etd.html)