Abstract
Because of its acute sensitivity to distance in the nanometer scale, Förster resonance energy transfer (FRET) has found a large variety of applications in many fields of chemistry, physics, and biology. One important issue regarding the correct usage of FRET is its dependence on the donor-acceptor relative orientation, expressed as the orientation factor κ2. Different donor/acceptor conformations can lead to κ2 values in the 0 ≤ κ2 ≤ 4 range. Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected in the quality of information that can be retrieved from a FRET experiment. In most cases, the average value of κ2 corresponding to the dynamic isotropic limit (<κ2> = 2/3) is used for computation of R0 and hence donor-acceptor distances and acceptor concentrations. However, this can lead to significant error in unfavorable cases. This issue is more critical in membrane systems, because of their intrinsically anisotropic nature and their reduced fluidity in comparison to most common solvents. Here, a simple numerical simulation method for estimation of the probability density function of κ2 for membrane-embedded donor and acceptor fluorophores in the dynamic regime is presented. In the simplest form, the proposed procedure uses as input the most probable orientations of the donor and acceptor transition dipoles, obtained by experimental (including linear dichroism) or theoretical (such as molecular dynamics simulation) techniques. Optionally, information about the widths of the donor and/or acceptor angular distributions may be incorporated. The methodology is illustrated for special limiting cases and common membrane FRET pairs.
Highlights
Fluorescence spectroscopy has been utilized as a major tool in biophysics in general, and membrane studies in particular, for decades
One parameter for which there is no experimental technique suited to a definite measurement is the Förster resonance energy transfer (FRET) orientation factor, κ2
Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected on the quality of information that can be retrieved from a FRET experiment
Summary
Fluorescence spectroscopy has been utilized as a major tool in biophysics in general, and membrane studies in particular, for decades. Quantitative applications of membrane fluorescence studies to the recovery of structural and partition information frequently require, as input, results obtained from other techniques. Some of these can be measured experimentally, such as area/lipid, lipid molar volume or bilayer thickness. Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected on the quality of information that can be retrieved from a FRET experiment. The theoretical value for the so-called dynamic isotropic limit ( = 2/3) is used, but uncertainty is still widely regarded as an inconvenience [5] that may be especially important in membranes, because of their intrinsic anisotropic nature and the restricted rotational mobility experienced by fluorophores incorporated inside the bilayer. The 2/3 value corresponds to the dynamic limit, where rotation of both donor and acceptor is fast compared with the excited state lifetime
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