Today’s take-home lessons: FRET (i. e Today’s take-home lessons: FRET (i.e. what you should be able to answer at end of lecture) How to measure: Donor Intensity, Lifetime Acceptor Intensity (very good way but more difficult) Why is E.T. a to (R/Ro)-6 [it depends on the near-field component of the electric field, which goes like R-3 with some normalization constant Ro-3

FRET: measuring conformational changes of biomolecules FRET useful for 20-80Å Distance dependent interactions between green and red light bulbs can be used to deduce the shape of the scissors during the function.

Fluorescence Resonance Energy Transfer (FRET) Spectroscopic Ruler for measuring nm-scale distances, binding R (Å) E Ro  50 Å Energy Transfer Donor Acceptor Dipole-Dipole distant-dependent energy transfer Time Time Look at relative amounts of green & red

Donor Intensity or Donor Lifetime FRET Measured by Donor Intensity or Donor Lifetime Call the Donor Intensity ID , Lifetime tD Donor Intensity in the presence of Acceptor IDA , tDA E = 1 – IDA/ID E = 1 – tDA/tD In FRET, there is a drop in donor’s intensity and lifetime k = krad + kn.r. tD = 1/k = trad + tn.r. QY = krad/(krad + kn.r) k = krad + kn.r. + kFRET tDA = 1/k = trad + tn.r + tFRET QY = krad/(krad + kn.r + kFRET)

Example of FRET What is the end-to-end distance on a dsDNA? D= Fluorescein A = Tetramethylrhodamine

Example of FRET (on DNA) E.T. by decreases in donor emission. Need to compare two samples, d-only, and D-A. Where are the donors intensity, and excited state lifetime in the presence of acceptor; without the acceptor. Donor quenching Acceptor “sensitized” emission The more Energy Transfer (donor and acceptor closer together)… more donor decreases, more the acceptor increases.

Example of FRET (on DNA) Donor quenching Acceptor “sensitized” emission What if the acceptor doesn’t fluoresce? Is E=0? (It has a quantum yield of fluorescence = 0) But it accepts just fine (Absorption is normal) No! Sensitized emission = 0, but donor quenching still happens

Example of FRET (on DNA) Measured w Acceptor’s Sensitized Emission E.T. by increase in acceptor fluorescence and compare it to residual donor emission. Need to compare one sample at two l and also measure their quantum yields. Ideal case, where direct acceptor’s emission = 0 (Next slide, subtract it off) Donor quenching Acceptor “sensitized” emission Energy Transfer (E) is an indication of excitations, not emission. Must divide by quantum yields.

Example of FRET (on DNA) Measured w Acceptor’s Sensitized Emission Real case, where direct acceptor’s emission ≠ 0. How to subtract it off? Donor quenching Acceptor “sensitized” emission [Below not discussed in lecture] Use two lasers, one to induce FRET, the other where only the acceptor excites. (e.g. 488 nm for Fl: 532 nm for TMR: see next slide) Then take an acceptor-only sample, excite at the two wavelengths and multiply by the ratio of the acceptors absorption at these two wavelengths.

Why does E.T. go like (R/Ro)-6

E.T. a 1/R6 :How is kET dependent on R? How does the energy go with distance? Think of a source emitting and look at R1, then 2R1, 3R1. U ≈1/R2 (Traveling: photons) How does electric field go like? U ≈ E2 ; E ≈ 1/R This is in the “Far-field”: (d >> l) In the Near-field (d << l) (must remember your E&M) E ≈1/R3 peE = pe/R3 Dipole emitting: Energy = U = Dipole absorption: paE Probability that absorbing molecule (dipole) absorbs the light So light emission goes like paE x peE = papeE2, pepa/R6 ≈ E.T. So (classically) E.T. goes like R-6 and pepa ~ Ro6 (a constant with right units) E.T. =1/(1 +knd/kET) = 1/(1 + (R6/Ro6)) = 1/(1 + 1/kETtD)