Biological Nanomachines

Transcription

1 Biological Nanomachines Yann R Chemla Dept. of Physics, University of Illinois at Urbana Champaign Saturday Physics for Everyone, Sept. 14, 2013

2 Biophysics at Illinois

3 Part I: WHAT IS BIOPHYSICS?

4 Physicists doing biology? Q: Why are physicists interested in biology? A: Physicists want to understand the world around us DNA E. coli cells A cat Simple Complex

5 What is biophysics? Applying techniques or ideas from physics to biological problems Central problems in biology will become accessible to analysis through basic physical laws. A biophysicist s description of biological phenomena aims to be: Quantitative (mathematical) Simple (captures enough detail but not too much) General (applicable to more than one system) Physics in a New Era NRC(2001) Grand challenges: Applying Physics to Biology Understanding Complex Systems

6 Part II: MOLECULAR NANOMACHINES

7 The cellular factory The cell =a nano scale factory of molecular machines DNA genetic blueprint to allcomponents of the cell Proteins carry out cellular tasks Molecular machine Drawing courtesy of M. Spies

8 Molecular machines Molecular machines move cargo around the cell inner life of the cell/

9 Molecular machines Molecular machines copy the cell s DNA mechanism of replication basic.html

10 Molecular machines Molecular machines propel cells

11 Molecular distances Strand of hair Bacterial cell (E. coli) > > 01mm micron = 1/100 X Strand of DNA 1 nanometer = 1/100,000 X Limit of light microscope

12 Molecular forces Weight of a small apple 1 Newton > Force exerted by a molecular nanomachine 1 piconewton (pn) = 1/1,000,000,000,000 X The apple that fell on Newton s head

13 Molecular energies Gallon of gasoline Calories in apple 100 MegaJoules 50 Calories = 1/1,000 X ATP: fuel of the cell > > 100 pn nm = 1/100,000,000,000,000, 000,000,000,000 X Adenosine Diphosphate Triphosphate (ADP) (ATP) + Phosphate (P i )

14 Measurement How do you measure anything? Too small to see Forces & energies too small to detect Traditional biochemistry Test lots of molecules together in a test tube

15 Bulk biochemistry Individual proteins move stochastically (= at random) This is a problem when doing traditional bulk biochemistry This one went slow Ideally, we want to study these This one one molecule paused at a time This one stopped START FINISH

16 Measurement How do you measure anything? Too small to see Forces & energies too small to detect Single molecule techniques to the rescue! 280 center Photons width 250 nm 80 Optical traps Y axis Sensitive to individual molecules! X Data Single molecule fluorescence

17 Part II: SINGLE MOLECULE TECHNIQUES

18 Optical tweezers Gradient force: F = (p E) = α E 2 Linear spring K ~ 0.1pN/nm measure pn, nm Ashkin et al., Opt. Lett. 11, 288 (1986)

19 The optical trap......a really expensive LEGO set 19

20 High resolution traps Optical tweezers can access Ångstrom length scales! 1 Ångstrom = 1/10 nanometer CAGT... Temperature controlled, noise free environment GTCA... 1 DNA basepair = 3.4Å 1Å 59 Loomis

21 Typical geometries Typical trap experiments involve tethering a single molecule & detecting changes to its length: Surface based (kinesin) Visscher et al. Nature (1999) But tthere are many others...

22 Example experiment Stretching a DNA hairpin : 3 biotin 3 digoxigenin Hairpin DNA handle DNA handle Streptavidin bead Anti digoxigenin bead Hairpin see also: Woodside et al., PNAS (2006)

23 Traps in action

24 Gone fishing 24

25 DNA Hairpin Transition Force ~15 pn Red = stretching Green = relaxing Hairpin protocol: Woodside et al., PNAS (2006) 25

26 Part III: DNA MOTORS

27 DNA Helicases Helicases unwind the strands of DNA Fuel: ATP ADP + P i Uses ATP fuel to move on one strand of DNA Unwinds double stranded DNA ahead Critical role in replication, recombination and repair 3 Review of helicase: Lohman et al., Nat. Rev. Mol. Cell Biol

28 Repair helicase Helicases are involved in repairing damaged DNA XPDhelicase atomic structure XPB TFIIH RPA XPD Fan et al., Cell (2008) Nucleotide excision repair

29 Hairpin Assay Monitor unwinding of a DNA hi hairpin i (under constant force) Δx Change (Δx) in tether extension reveals unwinding activity Qi et al., elife 2013

30 XPD Stepping Dynamics AT TA 6 10 AT GC 9 7 TA 8 CG GC GC DNA sequence affects reversals Time (s) Average step size is 1 bp Backstepping is frequent Step size (bp) Qi et al. elife (2013) Step finding: Kerssemakers et al., Nature (2006)

31 Helicase mechanism Conclusions XPD unwinds 1 bp at a time 2. Unwinds & slips DNA repetitively 3. Stalling & backstepping related to DNA sequence New model 1. Helicase unwinds by passive mechanism 2. Repetitive mechanism related to role in repair ATP 3 ADP + P i 5 31

32 Fishing... in the dark Wouldn t it be nice to see what s on the fishing line? 32

33 Single molecule fluorescence It is possible to see measure light from distances a single with molecule! pairs of fluorescent molecules FRET Photons Spectroscopic ruler Y axis X Data 5 0 Courtesy of Paul Selvin Roy, Hohng & Ha, Nat. Meth. (2008)

34 Fluorescence & Trap Bead 1 Bead 2 Photo ons/s 1 μm Fluor. Comstock et al., Nat. Meth. (2011) 34

35 UvrD 2B domain orientation Open ON moving? 2B ~160 rotation Closed Off stalled? 2B Low FRET High FRET UvrD helicase atomic structure Conformation switches function? Crystal structure: Closed at junction (presumed unwinding). Biochemistry and single molecule: Open during motion. Closed at junction stalled. FRET + Trap Jia, Lohman et al., JMB Park, Ha et al., Cell Lee and Yang, Cell Singleton et al., Ann. Rev. Biochem

36 Measuring UvrD conformation Dual labeled UvrD DNA Unwo ound (bp) Photo ons (khz) I Donor 0 I Acceptor FR RET Efficien cy Comstock et al., in preparation 1 Closed 0.5 Open Time (s) 36

37 Conformation & directionality Velociity (bp/s) Correlation between: conformation (closed/open) & directionality (unwinding/annealing) FRET Comstock et al., in preparation

38 Strand reversal? Conclusions 1. Closed when unwinding hairpin 2. Open when reannealing Open 3 5 Closed New model B domain remains anchored to dsdna 2. Motor domains switch strands* Open Closed 3 *Dessinges, et. al., PNAS

39 Part IV: OUTLOOK

40 Biophysicists wear many hats... Molecular biology Nature of research Experimentalist build instruments Biologist develop the biological system Theorist model the data Optics Data collection and analysis

41 Take home message Hey You put physics into my biology! No You put biology into my physics! BIOLOGY PHYSICS Quantification of Biological Systems TAKE HOME MESSAGE These advances present new directions for BOTH biology and physics. 41

42 Acknowledgements XPD Helicase stepping Zhi Qi Maria Spies & Robert Pugh (Univ. of Iowa) UvrD trap & fluorescence: Matt Comstock* Kevin Whitley Taekjip Ha (Univ. of Illinois) Tim Lohman & Haifeng Jia (Washington Univ.) Now at: Columbia Univ. * Michigan State Univ. Funding: