Lecture 7: Fluorescence: Polarization and FRET Bioc 5085 March 31, 2014

Flourescence Resonance Energy Transfer (FRET) 1 = Absorbance 2 = Emission 3 = Absorbance 4 = Emission D = Donor A = Acceptor FRET occurs and can be readily measured when: 1. Donor (D) has a high quantum yield (  f ) 2. Donor emission and acceptor (A) absorption spectra overlap (designed by J DA ) 3. Donor-acceptor distances are < 1.5 R o (R o is known as the Förster distance, see below)

Förster Theory and Förster Distances (R o ) Förster Distances = Distance between the donor and acceptor at which energy transfer is (on average) 50% efficient. According to Förster theory: 8.79 x is a combination of several physical constants  2 is the Förster orientation factor  is the refractive index of the medium (typically 1.4 for proteins)  d is the donor fluorescence quantum yield J DA is the Förster overlap integral All of the above can be determined experimentally, except for     is indeterminate since it depends not only on the orientation of the donor and acceptor dipoles, but also on the dynamics of these relative to one another   is theoretically 2/3 when the donor and acceptor fluorophores reorient isotropically relative to one another; this has been found to realized in most cases for probes that are not greatly restricted

FRET transfer efficiencies can be used to measure distances Importance of FRET to biochemistry is that the transfer efficiency, E, is a function of the separation of the fluors. Together with the known R o, E can be used to measure molecular distances:

Measurement of Transfer Efficiencies Measure Donor Emission Intensity ( 2 ) in the Presence and Absence of the Acceptor

FRET Donor-Acceptor Pairs: Trp as a Donor Wu & Brand, Anal. Biochem., 218, 1-13 (1994)

FRET Donor-Acceptor Pairs: “Attached” Donors and Acceptors Wu & Brand, Anal. Biochem., 218, 1-13 (1994)

Applications of FRET Wu & Brand, Anal. Biochem., 218, 1-13 (1994)

Fluorescence Polarization (FP) Polarizers transmit light that is either plane polarized along y or z (can usually be adjusted back-and-forth between these two positions).

Fluorescence Polarization (FP) Excitation Absorption Dipole I // II P //000  01 FP is based on selectively exciting molecules with their absorption transition moments (or equivalently absorption dipole) aligned parallel to the electric vector of polarized light (known as photoselection) Source “parallel” polarizer Absorption Transition Moment Angle Between Excitation & Emission Dipoles  0°  //101  000 0° // 0° Detect

Limiting Values of FP (+1 and -1) will never be obtained experimentally: 1)Assumed all dipoles in sample are identically aligned (not going to be true for liquid samples). 2) Assumed all molecules are fixed (not going to be true for liquid samples). 3)Assumed that the absorption and emission dipoles within fluorophore are collinear (not generally true). Factors that determine the extent of FP Limiting polarization (P o ) for molecules tumbling (isotropically) in solution is given by the Perrin-Weber Equation (addresses assumptions 1 and 3):  = Angle between absorption and emission dipoles

Extent to which molecule reorients relative to its fluorescence lifetime determines the extent of polarization (addresses second assumption)  = excited state lifetime  = rotational correlation time (i.e. rotational diffusion constant)  = solvent viscosity, V = volume of the fluorophore R = gas constant, T = temperature P  1/  hence, decreasing  will yield increased P P   hence, increasing  will yield increased P (   V and V  MW (by Stokes Law), hence P  MW)

Factors that effect extent of reorientation (and hence, extent of polarization) Figure 2. Simulation of the relationship between molecular weight (MW) and fluorescence polarization (P). Simulations are shown for dyes with various fluorescence lifetimes (  ): 1 ns (cyanine dyes) in purple, 4 ns (fluorescein and Alexa Fluor 488 dyes) in red, 6 ns (some BODIPY dyes) in green and 20 ns (dansyl dyes) in blue. At MW = 1000, P = for  = 1 ns, P = for  = 4 ns, P = for  = 6 ns and P = for  = 20 ns. Simulations assume P o (the fundamental polarization) = 0.5 and rigid attachment of dyes to spherical carriers. -Polarization increases as the MW increases (or as the solvent viscosity increases) -Polarization decreases as the excited state lifetime (  ) increases

FP Applications

Green Fluorescent Protein