Fluorescence Resonance Energy Transfer spectroscopy

FRET in our LAB

FRET in actin

Radial coordinate determinations in actin filaments

        Radial coordinate can be determined in actin filament (or other helically symmetrical structures) using fluorescence resonance energy transfer spectroscopy. A solution of actin monomers labelled with donor probes is mixed with a solution of actin monomers labelled at the same site with acceptor probes in order to obtain solutions with different acceptor molar ratio (acceptor concentration/ total protein concentration).
 
        FRET in actin filament can occur between a donor-labelled monomer  (square on n = 0) and the four adjacent monomers when labelled with acceptor probes (circles on n = -2, -1, +1, +2). Monomers above or below these four adjacent monomers do not significantly participate in the FRET efficiency because the distance is larger than 100 Å. The distance separating the donor and acceptor probe can be calculated knowing the Förster distance (R0) from the efficiency of transfer E:
       (1)

        R0 is the donor-acceptor distance where the efficiency of energy transfer is 50%. This distance is function of the probe pair. However, Eq. 1 is only valid when the distance is measured between a donor and an acceptor probe.
        In a filament, there can be up to four acceptors for one donor probe and the FRET efficiency for each arrangement of acceptors around a donor is given by:

                 (2)

where k is one of the 16 possible arrangements of acceptors around a donor, N is the number of acceptors for the kth arrangement and n is the position of the acceptor labelled monomers (n = -2, -1, +1, +2) in relation with the donor-labelled monomer (n = 0).
        Then, the FRET efficiency in a filament Et is the sum of the efficiency of transfer for each kth arrangement Ek multiplied by its probability dk. The sum is taken over all possible arrangements (K) and is given by:

        (3)
 

        Our Freeware FretLab obtains the efficiency of transfer from the donor probe lifetime and the donor lifetime in presence of acceptor probes and calculate the radius (r) corresponding using:

Rn = [ 2r2 (1 - cos(nf)) + (nT)2]1/2             (4)

where Rn is the distance between the donor and the acceptor located on monomer n = -2, -1, +1, +2; f is the rotation of the monomer in the genetic helix for F-actin f = -166.54° and T is the translation of the monomer in the filament and is equal to 27.5 Å in F-actin.
    The radius is calculated using a minimisation routine based on the golden section method. Once multiple radii have been obtained for different acceptor molar ratios, the program allows you to calculate the mean radius and its standard deviation.
 
 

Structure of the probes

The structure of these probes have been obtained using Cerius2 software in VISLAB and have been used to evaluate their maximum cords.
[Back to Top] [Back to Home Page]
This page was created by Pierre Moens