Today’s take-home lessons (i.e. what you should be able to answer at end of lecture)

1. Today’s Announcements Today’s take-home lessons(i.e. what you should be able to answer at end of lecture) • Homework assigned #6 due next Monday, 4/12 in class. • Next Monday 4/12: Klaus Schulten birds and magnetotaxis. • Next Wednesday 4/14: VMD (computer analysis) FRET – why it’s useful, R-6 dependence; R0 (3-7 nm), very convenient.

2. Quiz 1. Two models of DNA: A ________________________model assumes that DNA chains are completely straight, unstretchable. No thermal fluctuations away from straight line are allowed. The polymer can only disorder at the joints between segments. A ______________________model assumes that DNA chains have a correlation length called the persistence length. Freely jointed chain (FJC) Worm-like chain (WLC) 2. DNA has both twist and writhe. What is the twist and writhe number in the follow graph? Left:_______ ________, Center: ____ _________, Right: _____ _________ Tw = 0, Wr = 0 Tw = 2, Wr = 0 Tw = 0, Wr = 2 3. Resolution of a microscope is (classically) limited by Heisenberg uncertainty principle and is equal to __________________. λ/2 N.A 4. If you excite a fluorescent molecule with light of a certain wavelength, the molecule will emit light at a ______________ wavelength. longer 5. There are three families of motor proteins called: _______ ______ and _______. 6. List two advantages of two-photon microscopy: ______________________ and ______________________________. Kinesin, Myosin, Dynein Low light scattering Inherent z-resolution (or confocality)

3. FRET: measuring conformational changes of single biomolecules FRET FRET useful for 20-80Å The distance 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

5. Stokes Shift (10-100 nm) Absorption Fluorescence Wavelength Reminder: Fluorescence properties. Excitation from lowest ground state to excited states (fsec). Relaxation to lowest excitation singlet state (psec). Transition to multiple ground states (nsec).

6. Lifetimes and Quantum Yields Lifetime (td): exp(-t/td): 1-100nsec td = 1/k Quantum yield(f) : krad/ (krad +knon-rad) [how much of excited-state energy goes into light vs. heat] td, k

7. 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

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

9. Classically: How is kET dependent on R? How does electric field go like? Far-field: Near-field: 1/R2 1/R3 (d << l) peE = pe/R3 (E = electric field) Dipole emitting: Energy = U = Dipole absorption: paE Probability that absorbing molecule (dipole) absorbs the light So light absorbed goes like: paE x peE = papeE2 = pepa/R6 E.T. = 1/(1+ 1/kETtD) E.T. = 1/(1+ knd/kET) E.T. = 1/(1+ (R6/Ro6)) Classically: E.T. goes like R-6 , Depends on Ro

10. Quantum Mechanically Determine Hamiltonian (Energy) of Interaction. By Fermi’s Golden Rule, rate goes like H2. Dipoles interacting: Dipolar Interaction depends on R3: E.T. = H2 = R6

11. Terms in Ro in Angstroms where J is the normalized spectral overlap of the donor emission (fD) and acceptor absorption (eA) (Draw out ea(l)and fd(l) and show how you calculate J.)

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

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

14. Spectral Overlap terms Spectral Overlap between Donor & Acceptor Emission (J) For CFP and YFP, Ro, or Förster radius, is 49-52Å. http://mekentosj.com/science/fret/

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

16. 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.

17. y qD D qDA qA R • k2 is usually not known and is assumed to have a value of 2/3 (Randomized distribution) A z x • This assumption assumes D and A probes exhibit a high degree of rotational motion k2 : Orientation Factor The spatial relationship between the DONOR emission dipole moment and the ACCEPTOR absorption dipole moment (0< k2 >4) k2 often= 2/3 where qDA is the angle between the donor and acceptor transition dipole moments, qD (qA) is the angle between the donor (acceptor) transition dipole moment and the R vector joining the two dyes. k2 ranges from 0 if all angles are 90°, to 4 if all angles are 0°, and equals 2/3 if the donor and acceptor rapidly and completely rotate during the donor excited state lifetime.

18. Orientation of transition moments of cyanine fluorophores terminally attached to double-stranded DNA. Iqbal A et al. PNAS 2008;105:11176-11181

19. Simulation of the dependence of calculated efficiency of energy transfer between Cy3 and Cy5 terminally attached to duplex DNA as a function of the length of the helix. Iqbal A et al. PNAS 2008;105:11176-11181

20. Efficiency of energy transfer for Cy3, Cy5-labeled DNA duplexes as a function of duplex length. Orientation Effect observed, verified! Iqbal A et al. PNAS 2008;105:11176-11181

21. Class evaluation 1. 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.