Resonance energy transfer in cells: a new look at fixation effect and receptor aggregation on cell membrane

Abstract

Fluorescence resonance energy transfer (FRET) measurements offer a reliable and noninvasive approach to studying protein and lipid colocalization in cells. We have considered systems in which FRET occurs as intramolecular and/or intermolecular process. The proposed dynamic FRET model shows that in the case of intermolecular process the degree of aggregation only slightly affects the energy transfer efficiency. The theory was tested on a set of donor-acceptor pairs in which energy transfer occurs intramolecularly, intermolecularly, or both. The obtained experimental results are in a good agreement with the proposed model. It is well known that the energy transfer efficiency depends both on the distance between the donor and acceptor molecules and the relative orientation of their respective transition dipole moments. This dual dependence often leads to ambiguity. In this article, we show how FRET efficiency can be significantly reduced even in highly coupled system through conformational restrictions in the donor-acceptor pair. Importantly, such restrictions can be imposed on the system by cell fixation, a procedure routinely used when conducting FRET measurements.

FIGURE 1

Schematic representation of the systems in which intermolecular (A), both inter- and intramolecular (B), and intermolecular-only (C) energy transfers occur.

FIGURE 2

Dependence of intermolecular FRET efficiency on degree of aggregation. The curves were generated using Eq. 10 for equal concentrations of donor and acceptor and different values of and (squares); (diamonds); (triangles); (circles); and (crosses). The solid curves were calculated using and values from the experimental FRET data in Table 3 for the SE method. The upper curve corresponds to the CFP-YFP coupled and uncoupled systems (); the bottom curve corresponds to the YFP-CFP coupled and uncoupled systems ().

FIGURE 3

Correction curve used to evaluate the amount of donor bleedthrough into the acceptor channel.

FIGURE 4

HEK 293 cells transfected with CFP-YFP fusion protein. Panels A and C represent CFP channel before and after bleaching; panels B and D represent YFP channel before and after bleaching. The acceptor was bleached out of cell 1, and cell 2 was used as a control.

FIGURE 5

Dark-shaded and light-shaded planes represent the planes where transitional dipole moments of the donor and acceptor are located. The values θ_DA, θ_D, and θ_A are polar angles used to define relative orientation of the donor and acceptor and calculate the orientation factor κ.