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

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. HDTRA1-09-1-008

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

Introduction to Protein-DNA

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

Protein is an amino-acid polymer folds into different structures

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

TUS is one Protein we might study with our UK collaborator EColi replication termination protein from Protein Data Bank http://www.pdb.org/pdb/explore/jmol.do?structureId=2I05&bionumber=1

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

Introduction to Optical Tweezers

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

The Parameters we calibration Z 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

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

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.

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.

Feedback control

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

Optical tweezers are really sensitive to air turbulences

We use He-Ne to align IR

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

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

Unzipping DNA

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)

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

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

; 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.

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

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

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

Preliminary Data & Plans

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

Preliminary data dsDNA overstretching force is around 60pN

ds DNA unzipping force is 14 to 18 pN without protein

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

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

Thank You