Validation of depth-dependent fluorescence quenching in membranes by molecular dynamics simulation of tryptophan...

Depth-dependent fluorescence quenching is an important tool for studying the penetration of proteins and peptides into lipid bilayers. Extracting quantitative information from quenching data is, however, complicated by (1) a limited number of experimentally available quenchers and (2) thermal disorder resulting in broad distributions of the transverse positions of both quenchers and fluorophores. Here we validate and refine a general approach to determining the location of a fluorescent probe along the bilayer normal from quenching data, based on a molecular dynamics (MD) simulation of a model compound, tryptophan octyl ester (TOE), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. The TOE ring was found to lie deeply within the bilayer (most probable position of 13.3 Å and center-of-weight of the distribution of 14.8 Å from the bilayer center), and it was very broadly distributed (with 9 Å depth distribution width), which is consistent with previous experimental observations. The depth-dependent quenching profiles were simulated by treating carbon atoms of the lipid acyl chain of POPC as "pseudo-quenchers" and calculating appropriate transverse overlaps and collision rates with indole atoms of TOE. These simulated quenching profiles were well fitted by a Gaussian function of depth, as is routinely done with experimental data subjected to the distribution analysis procedure [Methods Enzymol. 1997, 278, 462-473]. Comparison of the collisional pseudoquenching profiles with the actual profiles of the indole moiety of TOE allows for testing of the validity of the data analysis and identification of the possible sources of error in calculating depths of membrane penetration from quenching data.