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Notes about these slides

Notes about these slides. Slides presented at Physics 500 / 400 Seminar @ U. New Mexico, January 18, 2007 by Steve Koch.

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Notes about these slides

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  1. Notes about these slides • Slides presented at Physics 500 / 400 Seminar @ U. New Mexico, January 18, 2007 by Steve Koch. • I think I have attributed all images and data that aren’t from my own publications or work, but it’s possible I’ve missed something. You should probably check with me before propagating anything. In most cases I can give you original drawing files if you want. • As noted on the acknowledgements slides, this work is highly collaborative, thanks to everyone! • Slides are rough & subject to errors…please ask questions in the discussion forums and talk about things!

  2. Welcome to the Seminar on Biophysics and Medicine! • Demo course web page • Course requirements: • Show up and ask questions! • Grad students: 10 minute talk about research • How should we do online discussion forum? • Openwetware.org • Demo Pub Med / Bookshelf

  3. Studying protein-DNA interactions by unzipping single DNA molecules: What new information can we obtain? Steve Koch, January 18, 2007, Physics 500/400 SeminarAssistant Professor, Physics and Astronomy and CHTM University of New Mexico

  4. Outline 1. Single-molecule manipulation capabilities provide new biological information 2. Optical Tweezers: Unzipping DNA molecules to probe protein-DNA interactions 3. Magnetic Forces: Improved efficiency for DNA unzipping; eukaryotic RNA Polymerase; molecular motors 4. I am looking for graduate students!

  5. Thank you to my wonderful collaborators! Karen Adelman (NIH), Arthur La Porta (U. Maryland), Richard Yeh, Michelle D. Wang Gayle Thayer, Jim Martin, George Bachand, Alex Corwin, Maarten de Boer, Amanda Trent Peter Goodwin, Jim Werner, Dick Keller, Kim Rasmussen Funding

  6. DNA and proteins are structured polymers Hemoglobin Adenylatekinase Insulin Antibody Glutamine Synthetase Gareth White, molecular surface representations of various proteins Michael Ströck, ribbon / atomistic model Proteins are polymers of 20 amino acids Folding much more diverse than DNA More exposed chemical groups Many purposes in the cell, including structural, enzymatic, signaling DNA, polymer of 4 nucleotides, A,T,G, C [Adenine-Thymine] [Guanine-Cytosine] Typically the double-helical structure above, Watson-Crick base pairing Main purpose: storage of genetic information

  7. Proteins and DNA interact frequently in cells DNA is a polymer 2 nanometers wide (2 billionths of a meter) and up to 1 centimeter long! In eukaryotes, DNA is wound around histone proteins to form nucleosomes 3 billion basepair human genome 30,000 genes 12% encode DNA-binding proteins DNA-binding proteins critical forgene regulation Gene regulation crucial for cell behavior(All cell types in a human have thesame genome!) From Molecular Biology of the Cell (Pub Med online)

  8. http://opbs.okstate.edu/~petracek/Chapter%2026%20figures/Fig%2026-01a.JPGhttp://opbs.okstate.edu/~petracek/Chapter%2026%20figures/Fig%2026-01a.JPG http://www.uta.edu/biology/henry/classnotes/2457/ Why single-molecule biophysics? RNA Polymerase gives us one example Biophysics Problems in biology are fascinating! Also increasingly complex. Huge need for physics techniques (Instrumentation & Analysis) Transcription central to gene regulation

  9. Example: Single-molecule manipulation was used to discern the effect of a drug on RNA Polymerase Karen Adelman (Wang Lab), NIEHS Arthur La Porta (Wang Lab), U. Maryland Adelman et al. Mol Cell. 14, 753 (2004). Ensemble in vitro transcription assay Adelman et al. Mol Cell. 14, 753 (2004). (a drug) The “ensemble” assays show that the drug slows down transcription overall. But how? Slower catalysis? or Increased pausing? Gel electrophoresis

  10. Example: Single-molecule manipulation was used to discern the effect of a drug on RNA Polymerase Karen Adelman (Wang Lab), NIEHS Arthur La Porta (Wang Lab), U. Maryland Adelman et al. Mol Cell. 14, 753 (2004). Optical tweezers assay Video of a similar experiment from Berkeley http://alice.berkeley.edu/RNAP/ Adelman et al. Mol Cell. 14, 753 (2004). Can monitor the length of transcription in real-time Answer: The drug increases pausing of RNA Polymerase

  11. Using optical tweezers, we can apply and measure forces on single tethered biomolecules Optical Trap “Laser tweezers” Microsphere Biomolecular “Tether” Coverglass Wang Lab (Cornell) TweezersRichard Yeh Opportunities for MEMS and nanophotonics! Less costly, more accessible, more stable

  12. Using optical tweezers, we can apply and measure forces on single tethered biomolecules Quadrant photodiode to measure force Standard methods for attaching DNA to coverglass and bead Piezoelectric stage moves coverglass relative to trap center Dielectic particles (500 nm polystyrene) attracted to laser focus Microsphere Optical Trap Biomolecular “Tether” Coverglass piezoelectric stage Newton’s third law Force on bead = force on lasercollect exit light onto photodiodeto measure force, displacement Infrared laser focused through microscope objective

  13. Using optical tweezers, we can apply and measure forces on single tethered biomolecules Microsphere Biomolecular “Tether” Coverglass • Forces from < 1 pN to 100s pN • pN = piconewton, 1 trillionth of N • Length precision ~ 1 nm • Thermal energy • 4 pN – nm = 1/40 eV • Kinesin 8 nm step, 6 pN stall • (molecular motor) • RNA Polymerase 0.3 nm step, 25 pN stall • DNA Unzipping 15 pN

  14. E. Coli RNA Polymerase Transcription Karen Adelman et al. PNAS 2002, Mol. Cell 2004 Single Nucleosome Disruption Brent Brower-Toland, David Wacker et al. PNAS 2002, JMB 2005 DNA Unzipping with Bound Protein Koch et al. Biophys. J. 2002, Phys. Rev. Lett 2003 We built one versatile optical tweezers for use in several different biological systems Richard Yeh (Wang Lab), Bechtel-Nevada

  15. F F F single- stranded DNA F unzip zip double- stranded DNA F F DNA Unzipping: Mechanical force biases thermal opening / closing fluctuations Unzipping DNA first demonstrated: Bockelmann, Essevaz-Roulet, Heslot 1997 DNA Image: http://www.biophysics.org/btol/

  16. F F DNA Capped by hairpin (allows reversal) Characteristic Unzipping Force Plateau Force to unzip DNA depends on sequence This DNA Molecule has 17 nearly identical ~200 bp repeats

  17. DNA is a flexible polymer, subject to Brownian motion • Simulations

  18. F ssDNA Freely-Jointed Chain (Smith et al. 1996 Science) 0.80 nm persistence length 580 pN stretch modulus 0.54 nm contour length per nt Force Polymer Extension F j . . . 2 1 100 ms Polymer physics modeling lets us knowhow many bases pairs have been unzipped Statistical physics Velocity Clamp

  19. F ssDNA Freely-Jointed Chain (Smith et al. 1996 Science) 0.80 nm persistence length 580 pN stretch modulus 0.54 nm contour length per nt Force Polymer Extension F j . . . 2 1 100 ms Polymer physics modeling lets us knowhow many bases pairs have been unzipped Velocity Clamp unzip zip

  20. F F j . . . 2 1 Intuitively, one expects a binding protein to inhibit DNA unzipping Restriction enzymesBind and cut specific DNA sequencesWell-studied model system No Mg++ in binding buffer (High EDTA)prevents endonuclease activity. PDB: 1DC1 BsoBI dimer bound to DNA

  21. F F j . . . 2 1 Dramatic increase in unzipping force seen with700 pM BsoBI endonuclease

  22. F F j . . . 2 1 Dramatic increase in unzipping force seen with700 pM BsoBI endonuclease Very obvious increased force (Worked the first time!)

  23. F F j . . . 2 1 Dramatic increase in unzipping force seen with700 pM BsoBI endonuclease Very obvious increased force (Worked the first time!) Binding locations match predictions Arrows show unoccupied sites

  24. We have a new single molecule method for detecting where, when, and what of protein binding

  25. Detecting where a protein is bound allows single-molecule, ordered, reversible restriction mapping 1. Define threshold force 2. Unzip many molecules • Histogram data F > 20 pN(grayscale map)

  26. Three different restriction enzymes produce correct maps Binding detected where we expect; not where we don’t (Non-repetetive)

  27. “Traditional” Genome Mapping TechnologyHigh throughput restriction fingerprinting • Each lane is a separate BACHindIII digestion • Gels are digitized and then processed to find overlaps(Fingerprints remain unordered) • Project ramped up to about 20,000 fingerprint maps per week (about 1x coverage)(120 per hour) • Difficulties with small and closely spaced bands Source: Nature 2001 Genome Issue “A physical Map…”

  28. Slides after this we did not see today (1/18/07)

  29. New possibilities enabled due to ordered, non-catalytic, single-molecule method Repetitive DNA not a problem Can work with functional binding proteins(e.g. transcription factors) In principle could map a chromosome from single cell Microelectromechanical Systems (MEMS) Drawbacks Resolution decreases with length Not automated or easy yet!

  30. Detecting when a protein is bound permitssite-specific equilibrium constant measurement “When” = site-specific equilibrium association constant Protein + DNAsite proteinDNAsite Measure this ratio([protein] >> [DNA] for this assay)

  31. Method has been validated using well-studiedEcoRI – pBR322 DNA system Agreement in both magnitude and slopeIndicates 8 ion pairs involved in EcoRI-DNA binding Our method haslarger uncertaintyNeed for increased efficiency (Salt screens the electrostatic attraction of protein-DNA)

  32. There are many benefits of this site-specific, single-molecule equilibrium constant measurement Remove complication ofnon-specific DNAsituations with lower KA Can measure KA even when off-rate very highvery tricky with standard methods Probe multiple sequences simultaneously MSH2-MSH6 (mismatch repair protein) bindingaffinity, specificity, and ATP-dependent sliding Wang Lab: J. Jiang et al., Mol. Cell 20, 771 (2005)

  33. Analysis of forces can determine what is bound Forces = What / “how strong” 33 pN threshold correct 90% of time Can potentially distinguish binding species on a molecule by molecule basis Graph shows two different Protein-DNA complexes

  34. Magnetics and MEMS can provide complementary single-molecule capabilities, speedier results Koch, Thayer, Corwin, de Boer, APL 173901  Very compliant Microfabricated Spring Electromagnetic Force Apparatus

  35. Constructing electromagnetic “tweezers” for parallel single-molecule experiments Jim Martin, Gayle Thayer (Sandia) Peter Goodwin, Jim Werner, Dick Keller (LANL) Combination of proven SM technologies pN, nm sensitivity Many molecules in parallel Ideal for many experiments: protein – DNA / unzipping Short molecular bonds Transcription

  36. At Sandia / CINT, prototyped magnetic tweezers Fluorescence Microscopy ~ 1/10 millimeter Movie links probably won’t work. See “zero force epi.avi” and “700 fN epi” Zero Force 700 fN Force

  37. EWD Signal Current Frame number Proof of principle for instrument succeeded for 4400 basepair double-stranded DNA tethers Evanescent scattering signal cycling magnet, 1.5 micron dsDNA Movie links probably won’t work. See “TIR 1”

  38. 50 microns MEMS Force Sensor: A direct way of measuring forces on magnetic microspheres Alex Corwin, Maarten de Boer, Gayle Thayer (Sandia) Folded beam suspension As low as 0.1 pN / nm Differential Moire displacment sensing <1 pN sensitivity Standard processing (Sandia’s SUMMiT V™) Adjustable spring constant (dynamic maybe) Works in water (buffer) Insensitive to temperature or buffer conditions

  39. We can measure forces on single 3 micron beads to characterize their polydispersity 10 microns Microspheres glued with Micromanipulator Electromagnet pole Single microsphere Affix 2.8 micron bead to sensor Position bead relative to magnet pole Ramp current, measure displacement Remove bead, repeat with new bead

  40. We can measure forces on single 3 micron beads to characterize their polydispersity 10 microns Microspheres glued with Micromanipulator Electromagnet pole Single microsphere 9% s.d. in saturated moment of beads (Literature: 41% – 72%) This information critical for biophysics experiments

  41. We can also use a single bead as a micron-scale force sensor to map electromagnet force field Single bead affixed to edge of spring

  42. We can also use a single bead as a micron-scale force sensor to map electromagnet force field Single bead affixed to edge of spring Translate bead relative to magnet pole Use of simple spring provides force calibration, insensitive to: unknown magnetite content unknown electromagnet props. temperature, buffer, etc.

  43. We can also use a single bead as a micron-scale force sensor to map electromagnet force field Z= 160 m (Closest to pole) Z= 1000 m Good agreement with FEMM Calculations http://femm.foster-miller.net Absolute difference due to magnetite content and properties, etc. Results directly applicable to biophysics experiments using same bead / magnet system

  44. I hope I have shown the potential of single-molecule manipulation tools for biophysics experiments And we have only scratched the surface of what can be done! Your ideas can help!!!

  45. Thank you to my wonderful collaborators! Karen Adelman (NIH), Arthur La Porta (U. Maryland), Richard Yeh, Michelle D. Wang Gayle Thayer, Jim Martin, George Bachand, Alex Corwin, Maarten de Boer, Amanda Trent Peter Goodwin, Jim Werner, Dick Keller, Kim Rasmussen Funding

  46. Slides after this one are scratch work.

  47. Optical tweezers can apply and measure forces Collect laser light to measure force • Small dielectric particles (beads) are attracted to the brightest spot of the laser focus • Create single-molecule tethers by securing one end to the coverglass, other end to bead. • Apply forces by moving the coverglass away from the center of the laser • Collection of laser light after passing through bead and calibration allow determination of length of tether, and force applied (pN) • Thermal energy1 kBT ~ 4 pN•nm Microsphere Biomolecule “Tether” Optical Trap Force Coverglass piezoelectric stage Laser focused through microscope objective

  48. F F j . . . 2 1 Same DNA, now in the presence EcoRI • 80 pM EcoRI • Each repeat of the DNA has two EcoRI binding sites separated by 11 bp

  49. F F j . . . 2 1 Same DNA, now in the presence EcoRI • 80 pM EcoRI • Each repeat of the DNA has two EcoRI binding sites separated by 11 bp • Standard deviation of “event” ~ 3 nt • Shows that UFAPAcan get fairly good relative resolution. • Could have applications for probing larger protein-DNA complexes. E.g., nucleosomes, transcription PICs

  50. Using optical tweezers, we can apply and measure forces on single tethered biomolecules Quadrant photodiode to measure force Trap stiffness proportional to laser intensity Microsphere Optical Trap Biomolecular “Tether” Coverglass piezoelectric stage Infrared laser focused through microscope objective

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