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Next Monday: Diffusion This afternoon: guidance on your papers.

Today ’ s Announcements. Next Monday: Diffusion This afternoon: guidance on your papers. Next Wednesday and Friday, your lectures. Practice! Be specific—give example(s) of what your saying, and then give general message. By this Friday, I will have when you talk (Wed, Friday).

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Next Monday: Diffusion This afternoon: guidance on your papers.

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  1. Today’s Announcements • Next Monday: Diffusion • This afternoon: guidance on your papers. • Next Wednesday and Friday, your lectures. • Practice! • Be specific—give example(s) of what your saying, and then give general message. • By this Friday, I will have when you talk (Wed, Friday). • By 2 am on Wednesday April 17th, • or by 2 am Friday April 19th, • you MUST send me your powerpoint slides

  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. You can measure ensemble FRET and single-molecule FRET An example of DNA and a major killer, Hepatitis C.

  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. 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

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

  6. 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). • How do you measure this? • n is the index of refraction (1.33 for water). • 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. Compare to known standard. Varies from 0 to 4; usually = 2/3.

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

  8. 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.

  9. E.T. by decreases in donor emission. 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.

  10. Fluorescein [D] & Tetramethylrhodamine [A] Good E.T. Ro ≈ 50 Å Tetramethylrhodamine (TMR) Fluorescein Isothiocyanate (amine reactive)

  11. (Ensemble) FRET on DNA Fluorescein to Rhodamine A separate series of data with reversed strand labeling gives a similar curve, and the independent fits agree within +2 Å for all the values L, a, and d, and within ±15° for f. Notice A, D, separated by length L, but not on-axis! Clegg at al, PNAS, 1993

  12. Can follow basic Reaction:e.g. Melting Temperature As [NaCl] goes up/down, what happens to melting temperature? Clegg at al, PNAS, 1993

  13. Orientation of transition moments of cyanine fluorophores terminally attached to double-stranded DNA. Cy3  Cy5 Iqbal A et al. PNAS 2008;105:11176-11181

  14. 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

  15. 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

  16. Stairway to Unwinding Sua Myong* Michael Brunoϯ Anna M. Pyleϯ Taekjip Ha* * University of Illinois Ϯ Yale University

  17. About HCV NS3 helicase • 1. Hepatitis C Virus (HCV) is a deadly virus affecting 170 million people in the world, but no cure or vaccine. • 2. Non-Structural protein 3 (NS3) is essential for viral replication. • 3.NS3 iscomposed of serine protease and a helicase domain • 4. NS3 unwinds both RNA and DNA duplexes with 3’ overhang • 5. In vivo, NS3 may assist polymerase by resolving RNA secondary structures or displacing other proteins.

  18. NS3 unwinds DNA in discrete steps of about three base pairs (bp). Dwell time analysis indicated that about three hidden steps are required before a 3-bp step is taken.

  19. Steps (~11 bp) and substeps (2-5 bp) in NS3 catalyzed RNA unwinding under assisting force • Dumont, Cheng, Serebrov, Beran,Tinoco Jr., Pyle, Bustamante. • Nature (2006)

  20. Watching Helicase in Action!

  21. SIX steps in 18 bp unwinding [NS3]=25nM [ATP]=4mM Temp = 32oC ATP ATP

  22. More traces with Six steps [NS3]=25nM [ATP]=4mM Temp = 32oC tr1 tr2 tr3 1 2 3 4 5 6 tr4 tr5 tr6

  23. Is each step due to one rate limiting step i.e. one ATP hydrolysis? -> Build dwell time histograms

  24. k k k n irreversible steps: Here n = 3. 3 hidden steps involved with each 3 bp step Non-exponential dwell time histograms If the three base-pair steps were the smallest steps i.e. there were no hidden substeps within, the dwell time distribution will follow an exponential decay. Smallest substep = Single basepair ! dwell time (sec)

  25. A movie for how NS3 moves Inchworm model with 1 bp hidden steps making 3 bp “real” steps! (A nuclear-based motor protein We made a little movie out of our results and a bit of imagination. The two domains over the DNA move in inchworm manner, one base at a time per ATP while the domain below the DNA stays anchored to the DNA through its interaction, possibly the tryptophan residue. Eventually, enough tension builds and DNA is unzipped in a three base pairs burst.

  26. A model for how NS3 moves Myong et al, Science,2007

  27. 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.

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