Theory for establishing proximity relations in biological membranes by excitation energy transfer measurements

Abstract

In a previous publication (Shaklai et al., 1977a) the present author developed a theory for evaluating proximity relations and surface densities sigma in biological membranes by measurements of excitation energy transfer from a donor attached to a specific site of a membrane protein and an acceptor attached to a specific carbon on a membrane lipid. It was assumed that the protein and lipid are randomly distributed in the plane of the membrane and that the donor and acceptor groups are confined to different planes in the membrane separated by a distance Rp. In this article several aspects of the theory presented in the previous paper are clarified, especially noting that the previous theoretical expressions for the time-dependent and steady state fluorescence intensities assumed that the labeled protein molecule is cylindrically symmetric with the symmetry axis perpendicular to the plane of the membrane and that the donor is positioned on the symmetry axis of the protein. This assumption is also implicitly or explicitly made in subsequent formulations by other investigators. In this article we generalize the theory to include the case where the donor is not on the symmetry axis of the labeled protein. Equations for calculating the time-dependent and steady state fluorescence intensities for this more general case are presented, and methods for applying these theoretical expressions to the analysis of steady state fluorescence intensity data and evaluation of proximity parameters are discussed. It is also shown in this article that the linear relation l/lo = 1 + Kq sigma previously derived for simple analysis of excitation transfer data for the condition rc/Ro 1 can be modified to apply to almost all practical ranges of rc/Ro without much affecting its simplicity in the analysis of experimental data.

The Structural Organization of Cationic Lipid-DNA Complexes

“…Equation 6 is essentially equivalent to the Stern-Volmer relationship describing fluorescence quenching, with representing the two-dimensional concentration of quencher. It has been previously dem- (30,31,35). In the case where R 0 /L app Ͼ 0.6, a correction must be made that takes the following form.…”

“…where the correction factor, ␥, is interpolated from data presented in Table I of Yguerabide (30). Further verification of L can be obtained by comparison of donor quenching with biexponential approximations to theoretical curves derived by Wolber and Hudson (31).…”

Clustering of Class I HLA Oligomers with CD8 and TCR: Three-Dimensional Models Based on Fluorescence Resonance Energy Transfer and Crystallographic Data

“…Therefore, the efficiency of FRET in this study will also depend on the donor to acceptor ratio. In such a situation only, "closest approach distances of labeled epitopes from the membrane surface" (perpendicular to the cell membrane) can be estimated according to the theory and model described by Yguerabide (29,30). The use of this approach is justified by the observation that at the applied concentrations of the lipid probe the Stern-Volmer plots displaying I da /I d (ratio of donor intensities in the presence and absence of acceptor, respectively) vs the surface density of BODIPY-PC varied linearly.…”

“…In these curves, the fractional change in donor intensity, defined as E/(1 Ϫ E) ϭ I D /I DA Ϫ 1, with E the energy transfer efficiency and I D and I DA the donor intensities in the absence and presence of acceptor, respectively, was plotted against the surface density of BODIPY-PC. The slopes of the fitting straight lines give the K q quenching constants, from which the distances of closest approach RϭR 0 3/2 (/2K q ) 1/4 were computed (29,30). A value of 3.9 nm was used for the R 0 Förster critical distance based on the emission and absorption spectra of X-SF and BODIPY-PC (581/591), respectively.…”

“…The BODIPY-PC molecules, however, may form a multiple acceptor array around donor Fab-labeled HLA molecules in the plane of the membrane, depending on their concentration. Thus, in this study, the actual acceptor density may also largely influence the FRET efficiency (29,30). Therefore, in this case, FRET measurements were conducted at different donor/acceptor ratios (Fig.…”