Today’s Announcements1. Today: finish FRET

2. Diffusion(Why moving in a cell is like swimming in concrete.)

2.Your written Paper, due Nov. 21st.

Today’s take-home lessons: FRET

Quick review of FRET – Fluorescence Resonance Energy Transfer.

An example of DNA and a major killer, Hepatitis C. Mechanism—hidden sub-steps detected by FRET

Cool video(remember GFP?)

https://www.youtube.com/watch?v=

sXM_WmwT2v4

Green Fluorescent Protein: Genetically-encoded dye

Fluorescent protein from jelly fish

FRET

FRET: measuring conformational changes of (single) biomolecules

Distance dependent interactions between green and red light bulbs can be used to deduce the shape of the scissors during the function.

FRET useful for 20-80Å

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)

Fluorescence Resonance Energy Transfer (FRET)

EnergyTransfer

Donor Acceptor

Dipole-Dipole distant-dependentenergy transfer

AD

R0AD

D A

R (Å)0 25 50 75 100

0.0

0.2

0.4

0.6

0.8

1.0

E

Ro 50 Å

60 )/(1

1

RRE

Spectroscopic Ruler for measuring nm-scale distances, binding

Time

Time

Look at relative amounts of green & red

https://www.youtube.com/watch?v=pMH8zcWa7WA&list=PLEGwTYt9TZjkRBwbZxywmg536QRtRbh4V

FRET between two proteins, one labeled with the donor (CFP) and a one with the acceptor (YFP). A third protein

gets in between the donor and acceptor.

10 nm– no FRET: donor emission, very little acceptor emission

Protein is displaced; < 10nm FRET

A video of FRET

Start at 2:40pm

Can follow basic Reaction:e.g. Melting Temperature

Clegg at al, PNAS, 1993

As [NaCl] goes up/down, what happens to melting temperature?

Terms in Ro (homework details)

in Angstroms

• J is the normalized spectral overlap of the donor emission (fD) and acceptor absorption (A)

• 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).• is a geometric factor related to the relative orientation of the transition dipoles of the donor and acceptor and their relative orientation in space.

60 )/(1

1

RRE

Compare to known standard.

Varies from 0 to 4; usually = 2/3.

How to measure Energy TransferDonor intensity decrease, donor lifetime decrease,

acceptor increase.

E.T. by increase in acceptor fluorescence and compare it to

residual donor emission. Need to compare one sample at two and

also measure their quantum yields.

AD

R0AD

D A

Time

Time

E.T. by decreases in donor emission.Need to compare two samples,

d-only, and D-A.

Where are the donor’s intensity, and

excited state lifetime in the presence of acceptor, and

________ are the same but without the acceptor.

Orientation Factor (homework details)

where DA is the angle between the donor and acceptor transition dipole moments, D (A) is the angle between the donor (acceptor) transition dipole moment and the R vector joining the two dyes.

2 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.

x

y

z

D

AR A

DDA

This assumption assumes D and A probes exhibit a high degree of rotational motion

2 is usually not known and is assumed to have a value of 2/3 (Randomized distribution)

The spatial relationship between the DONOR emission dipole moment and the ACCEPTOR absorption dipole moment

(0< 2 >4)2 often = 2/3

Orientation of transition moments of cyanine fluorophores terminally attached to double-stranded DNA.

Iqbal A et al. PNAS 2008;105:11176-11181

Cy3 Cy5

(Ensemble) FRET on DNA

Fluorescein to Rhodamine

Clegg at al, PNAS, 1993

A separate series of data withreversed strand labeling gives a similar

curve, and the independentfits agree within +2 Å for all the values

L, a, and d, and within ±15° for .

Notice A, D, separated by length L,

but not on-axis!

Efficiency of energy transfer for Cy3, Cy5-labeled DNA duplexes as a function of duplex length.

Iqbal A et al. PNAS 2008;105:11176-11181Orientation Effect observed, verified!

Why is there a bump around 14 bp and 19 bp?

Sua Myong*Michael Brunoϯ

Anna M. Pyleϯ

Taekjip Ha*

* University of IllinoisϮ Yale University

About HCV NS3 helicase1. 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 is composed 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.

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.

Conclusion of FRET studies on NS3

Watching Helicase in Action!

Helicase

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0

FR

ET

time (sec)

0 5 10 15 20 25 30 35

0

500

1000

1500

2000 B C

Inte

nsi

ty (a

u)

ATP

5 10 15 20 25 30 35 400.0

0.2

0.4

0.6

0.8

1.0

FR

ET

time (sec)

5 10 15 20 25 30 35 40

0

500

1000

1500 B C

Inte

nsi

ty (a

u)

ATP

SIX steps in 18 bp unwinding [NS3]=25nM[ATP]=4mMTemp = 32oC

-2 0 2 4 6 8 10 12 14 160.0

0.2

0.4

0.6

0.8

1.0 D 5 point AA Smoothing of hel15tr244_D

-2 0 2 4 6 8 10 12 14 16 180.0

0.2

0.4

0.6

0.8

1.0 D 5 point AA Smoothing of hel15tr80_D

0 2 4 6 8 100.0

0.2

0.4

0.6

0.8

1.0 D 5 point AA Smoothing of hel15tr269_D

-2 0 2 4 6 8 10 12 14 16 18 200.0

0.2

0.4

0.6

0.8

1.0 D 5 point AA Smoothing of hel15tr289_D

-5 0 5 10 15 20 25 30 35 400.0

0.2

0.4

0.6

0.8

1.0 D 9 point AA Smoothing of hel15tr363_D

tr1 tr2 tr3

tr4 tr5 tr6

[NS3]=25nM[ATP]=4mMTemp = 32oC

12

34

5

6

More traces with Six steps

Myong, Ha, Science, 2007

(vary [ATP], °T, dsDNA = 9 bp)

Three steps in 9 bp unwinding

1 step per 3 base pairs (again)

Is each step due to one rate limiting stepi.e. one ATP hydrolysis?

-> Build dwell time histograms (just like with molecular motors, i.e. myosin, kinesin, F1Fo-ATPase

Non-exponential dwell time histograms

Smallest substep = Single basepair !

dwell time (sec)

k k k

n irreversible steps: Here n = 3. 3 hidden steps involved with each 3 bp step

ktn et 1

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.

18 bp DNA 9 bp DNA

See (from Myong Science, 2007:

http://www.sciencemag.org/content/suppl/2007/07/26/317.5837.513.DC1/1144130s1.mov

(Summarized on next page.)

A model for how NS3 moves

Myong et al, Science,2007

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.