1. Effect of water on protein-DNA interaction: investigation
by single-molecule unzipping
with optical tweezers
Pranav Rathi
OSE

2. Acknowledgments Collaborations Funding Dr. Larry Herskowitz
Dr. Andy Maloney
Anthony Salvagno
Physics Ph.D. Student
Dr. Steven Koch
Collaborations
Susan Atlas—Lead of the DTRA project UNM Physics / Cancer Center / Director of CARC
Haiqing Liu (G. Mantano lab)—Microdevice applications of kinesin
LANL & Center for Integrated Nanotechnology (CINT)
Funding
DTRA—DTRA CB Basic Research Program under Grant No. HDTRA

3. Outline
1. Introduction to Protein-DNA
2. Introduction to Optical Tweezers
3. Unzipping DNA
4. Preliminary Data & Plans

4. Introduction to Protein-DNA

5. Michael Ströck, ribbon / atomistic model
DNA is a polymer
2 nm wide
.338 nm
The back bone is
negatively charged
DNA, is double/single stranded polymer of 4nucleotides, A,T,G,C [Adenine-Thymine] [Guanine-Cytosine]
Main purpose: storage of genetic information
Michael Ströck, ribbon / atomistic model

6. Protein is an amino-acid polymer folds into different structures

7. Proteins nearly execute all cell functions
Proteins are the building blocks
of the cells
Sequencing of human genome
shows that ~15% of the ~30000
genes encode proteins that
bind to nucleic acids

8. TUS is one Protein we might study with our UK collaborator
EColi replication termination protein from Protein Data Bank

9. Site specific Protein-DNA interaction
A particular sequence of nucleotides on
DNA is called specific site
Site specificity means that protein binds
strongly at that site of DNA in comparison
to any where else on DNA
The bond strength can be defined with the
association constant, which depends on
accessibility, solute concentration, pH,
ionic-bond strength……..
Protein

10. Introduction to Optical Tweezers

11. Optical Tweezers are used to apply forces over nanometer scale on the order of piconewtons
Show animation

12. The Parameters we calibrationZ
Trap center
Kx is the stiffness in x direction
X is displacement of bead center
from the trap center
Ho is the distance between beam
Waist and the trap center. X Ho
Beam waist
Surface

13. Calibration of stiffness Kx
We use Brownian noise to map the stiffness
Equation of motion for trapped bead
Power spectrum
After Fourier transformation
Cutoff frequency ωc
ωc ~ 200 to 600 Hz ~ 15 to 40 pN
Explain the procedure

14. Calibration of displacement X (DOG)
Sensitivity Vs z-Piezo
DOG Z
100nm
Surface
We scan a stuck bead with the trap in transverse x-direction in 100nm
increment heights from the surface and record the x-signal at QPD
The resultant DOG profile is fit with 5th degree polynomial. In the plane of maximum sensitivity, bead center and trap center overlaps.

15. Trap center does not coincide with beam waist
Do the DOG for .5 micron beads by using
z-lens, and find out the maximum sensitivity
plane, record the z-lens position
Do the DOG for 1 micron bead and repeat the
same procedure
3. Subtract the two recorded z-lens position,
this will be equal to
4. This defines the relationship between z-lens
translation and trap center translation in z
direction
5. Write a Lab View code to find out the surface
and record the intensity
6. Do a power-spectrum by using z-piezo away
from the surface and record
7. Now move the z-piezo toward surface and do
power-spectrum until the cutoff frequency drops
by a factor~2.
8. Now we know that the trap center is ~1 bead
radius away
9. Match the surface brightness with recorded.

16. Feedback control

17. Optical Tweezers Optical-Setup
He-Ne laser
ND-hard drive filter system
Up-stream
mirrors
ND:YAG-laser
AOM
Slow-Shutter
Expansion Lens1
125mm
Fast-Shutter
Periscope
Expansion Lens 2
850mm
Down-stream
mirrors
ND-CD drive filter system
Steer lens (100mm)
Assembly
1:1 telescope
Oil immersion
1.4NACondenser
Sample plane
IR-filter
Dichroic
Fiber-bundle
Fiber
Illuminator
White light
Dichroic
ND filter
50mm
QPD
lens
IR-water immersion
1.2NA 60X Objective
Laser
line filter
QPD

18. Optical tweezers are really sensitive to air turbulences

19. We use He-Ne to align IR

20. Steer lens assembly is used to move trap in xy-sample plane

21. QPD is used to sense the bead-movement in sample plain

22. Unzipping DNA

23. with Freely Joined Chain and Worm Like Chain
Unzipping is mapped
with Freely Joined Chain and Worm Like Chain
FJC
WLC
Unzipping construct is a combination of anchor DNA and unzipping DNA. Anchor DNA is dsDNA. Unzipping DNA transforms into ssDNA while unzipping
The unzipped length is measure in terms of number of base-pairs unzipped (j)

24. Freely-Jointed Chain is used to model ssDNA
a is Kuhn length, Lo is contour length
Langevin function
Persistence length Lp=a/2
Extended FJC is used when external force is present
We use FJC to calculate the number of base pairs unzipped under the force F

25. Worm-Like Chain is used to model dsDNA
Persistence length
High force regime
From .1pN to 10pN
For < 10pN
This model is used when force is above .1pN

26. ; Protein-DNA bond strength is subject to applied external force
Dissociation Rate at F=0
At F≠0 ;
toff is bond dissociation life time under force
To characterize the bonding now our goal is to determine parameter d
and toff that will lead to barrier energy Eo and tD for particular buffer conditions.

27. Unbinding force probability density function is used to find
Parameters of the energy well
Most probable unbinding force F* is
given by the peak value of PDF

28. Water interaction with protein-DNA affects the protein-DNA interaction
Salt
In terms of chemical potential
Water movement
Higher chemical potential
Lower chemical potential

29. Water forms hydration layers around objects in water
Chemical potential of
hydration layers is higher
than the bulk water
Bulk water
Bulk chemical potential can be changed by introducing more solute to the
bulk water

30. Preliminary Data & Plans

31. DNA Tethering
Create flow cell from double stick tape, slide and cover slip Flow anti-dig, surface blocker, tethering DNA, microspheres, and wash sequentially

32. Preliminary data
dsDNA overstretching force is around 60pN

33. ds DNA unzipping force is 14 to 18 pN without protein

34. Expected experiments & Results
DNA unzipping with protein
Different
proteins
Probably
D2O
Different osmotic stress
DNA stretching with protein
Different
proteins
Probably
D2O
Different osmotic stress

35. Significance of results
Water is a part of all biological systems
Water has been ignored mostly in protein
-DNA studies, that’s why we want to
study it
Protein interaction with DNA in cells is
very important. Our results will help in
understanding this better and might also be useful for understanding other processes like replication-termination

36. Thank You