Refining Analysis of Membrane Penetration with Depth-Dependent Fluorescence Quenching and Molecular Dynamics Simulations

Abstract

Depth-dependent fluorescence quenching by lipid-attached quenchers is an important tool for determining the penetration of proteins and peptides into lipid bilayers. Extracting quantitative information and accurate calculations of the depth of the fluorophore is complicated by (1) lack of reliable experimental data for penetration depths of the quenchers and (2) thermal disorder resulting in broad distributions of the transverse positions of both quenchers and fluorophores. First, we validate and refine a general approach of Distribution Analysis (DA) to determining the depth location of a model fluorescent probe in POPC bilayer using molecular dynamics (MD) simulations. MD simulated depth-dependent quenching profiles of the probe estimated by treating carbon atoms of the lipid acyl chain as pseudo-quenchers were well characterized by a Gaussian function of depth, being in agreement with the main principle of DA. The transverse distribution of the probe estimated from these simulated quenching experiments corresponded well to the underlying molecular distribution of the probe. Second, the depth of Tempo-PC and a series of Doxyl-labeled lipids (n-Doxyl-PC), in which a spin moiety was covalently attached either to a headgroup or to n-th carbon atoms of the stearoyl chain of the host lipid (where n = 5, 7, 10, 12, and 14), was refined using MD simulation. The results of simulations were experimentally validated using quenching of a lipid-attached probe. We further improved the precision of depth determination by applying a new protocol based on a difference of steady-state and time-resolved quenching. The resulting static quenching profiles are less susceptible to broadening caused by diffusion occurring during excited-state lifetime. Finally, we illustrate the application of this methodology by determining membrane penetration of a series of site selectively-labeled mutants of diphtheria toxin T-domain. NIH-GM069783.