HW returned today. 2. Another HW assigned yesterday, because I messed up. Due Friday.

1. Today’s Announcements • HW returned today. • 2. Another HW assigned yesterday, because I messed up. Due Friday.

2. Today’s take-home lessons(i.e. what you should be able to answer at end of lecture) FRET – Fluorescence Resonance Energy Transfer (Invented in 1967) why its useful, R-6 dependence; R0 (2-8 nm), very convenient.

3. FRET: measuring conformational changes of (single) biomolecules FRET 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.

4. FRET is so useful because Ro (2-8 nm) is often ideal Bigger Ro (>8 nm) can use FIONA-type techniques (where you use two-different colored-labels)

5. Donor Emission Donor Emission http://mekentosj.com/science/fret/

6. Acceptor Emission Acceptor Emission http://mekentosj.com/science/fret/

7. E.T. leads to decrease in Donor Emission & Increase in Acceptor Emission http://mekentosj.com/science/fret/

8. E Energy Ro  50 Å Transfer Acceptor Donor R (Å) Dipole-dipole Distant-dependent Energy transfer Time Time Fluorescence Resonance Energy Transfer (FRET) Spectroscopic Ruler for measuring nm-scale distances, binding Look at relative amounts ofgreen& red

9. Energy Transfer Acceptor Donor Derivation of 1/R6 Energy Transfer. = function (kET, knd) E.T. = kET/(kET + knd) E.T. = 1/(1+ knd/kET) E.T. = 1/(1+ 1/kETtD) Kn.d. How is kET dependent on R? kET knon-distance = knd = kf + kheat= 1/tD= 1/ lifetime

10. Classically: 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) U ≈ E2 ; E ≈ 1/R How does electric field go like? 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. Classically: E.T. goes like R-6 (Depends on Ro) E.T. = 1/(1+ (R6/Ro6)) E.T. = 1/(1+ 1/kETtD)

11. or ? Energy Transfer goes like… Take limit…

12. Terms in Ro in Angstroms • J is the normalized spectral overlap of the donor emission (fD) and acceptor absorption (eA) • qD is the quantum efficiency (or quantum yield) for donor emission in the absence of acceptor (qD = number of photons emitted divided by number of photons absorbed). • n is the index of refraction (1.33 for water; 1.29 for many organic molecules). • k2 is a geometric factor related to the relative orientation of the transition dipoles of the donor and acceptor and their relative orientation in space.

13. Terms in RoJ: Does donor emit where acceptor absorbs? in Angstroms where J is the normalized spectral overlap of the donor emission (fD) and acceptor absorption (eA) Spectral Overlap between Donor (CFP) & Acceptor (YFP) Emission Ro≈ 49-52Å.

14. With a measureable E.T. signal E.T. leads to decrease in Donor Emission & Increase in Acceptor Emission http://mekentosj.com/science/fret/

15. E.T. by changes in donor. Need to compare two samples, d-only, and D-A. 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. Where are the donor’s intensity, and excited state lifetime in the presence of acceptor, and ________ are the same but without the acceptor. Time Time How to measure Energy TransferDonor intensity decrease, donor lifetime decrease, acceptor increase.

16. Class evaluation • What was the most interesting thing you learned in class today? • 2. What are you confused about? • 3. Related to today’s subject, what would you like to know more about? • 4. Any helpful comments. Answer, and turn in at the end of class.