1. Lecture 2 Single-Molecule Methods

2. Advantages of single-molecule experiments Observe heterogeneity: static (differences between molecules) dynamic (history of a single molecule) Observe single molecules in vitro and in vivo, in real time Measure positions with nanometre precision Measure separations with Å ngström precision No need to synchronize population to observe dynamic behaviour Can study transient events Can apply force to molecules Very small sample sizes

3. Particle tracking Direct observation of rotation of F 1 ATPase, Part 2 120° periodicity reflects symmetry of F 1. 90°, 30° substeps reveal mechanistic detail. 40nm gold bead greatly reduces drag bead position measured by dark field microscopy Yasuda, R. et al., Nature 410, 898 (2001)

4. Optical transitions between molecular electronic energy levels Absorbance Fluorescence emission Cy3 wavelength nm 440490540 590640 690 emission filter excitation filter or laser absorbed photon emitted photon (radiative decay) non-radiative decay Fluorescence microscopy

5. Total Internal Reflection Fluorescence (TIRF) microscopy glass cover slip objective lens transmitted ray evanescent field reflected ray (total internal reflection) immersion fluid specimen in water ray incident on glass- water interface beyond critical angle n1n1 n2n2 x y

6. Fili, N. et al. Nucl. Acids Res. 38, (2010) Helicase activity measured by TIRF microscopy helicase unwinds immobilized duplex fluorophore binds to single-stranded DNA pauses and bursts of activity Each spot corresponds to a single helicase molecule. Intensity increases as the helicase unwinds its DNA substrate.

7. Single-molecule enzymatic dynamics Lu, H.P. et al. Science 1998;282:1877 (1998) Single molecules of cholesterol oxidase immobilized in an agarose gel The enzyme is fluorescent during part of the catalytic cycle, so individual catalytic turnovers can be monitored. The enzyme operates stochastically, but displays static and dynamic inhomogeneity. Static inhomogeneity: histogram of on-times for a single molecule fits a single exponential, giving a well- defined rate constant,BUT different molecules have different rate constants. Dynamic inhomogeneity: autocorrelation function of on- times reveals memory effects with correlation time ~1s. onoff deviation of m th on- time from mean

8. fluorescent label hand over hand? inchworm? asymmetric half steps 23nm52nm74nm full step  hand over hand Microscopy with 1 nm resolution: stepping of myosin V A Yildiz et al. Science 300, 2061(2003)

9. Flors, C. et al. ChemPhysChem 10, 2201 (2009) Super-resolution imaging using switchable fluorophores 1 μm reconstructed super- resolution image wide field image DNA

10. Fluorescence Resonance Energy Transfer (FRET) (Förster Resonance Energy Transfer) Energy transfer efficiency: excitation of donor radiative decay of donor energy transfer to acceptor radiative decay of acceptor donoracceptor r

11. Following dynamics and function of single molecules by FRET X Zhuang et al. Science 296,1473 (2002) substrate S docking interface cleavage site acceptor donor

12. Optical tweezers principle of operation

13. Power stroke of myosin II time displacement power stroke 5 nm Veigel, C. et al, Nature 398, 530 (1999)

14. Force exerted by kinesin Feedback maintains constant bead position in trap  constant force 8nm steps stall force Visscher, K. et al, Nature 400, 184 (1999)

15. Atomic Force Microscopy (AFM) High-resolution AFM images of native membrane proteins. (a) Ion-driven rotors from spinach chloroplast (b) I. tartaricus F o F 1 -ATP synthase (c) Native photosynthetic membrane from R. photometricum. (d) Perfringolysin O pore complexes. (e) Dimeric bovine rhodopsin. (f) Extracellular surface of gap junction hemichannels. Muller, D.J. & Engel, A. Nature Protocols 2, 2191 (2007) quadrant photodiode cantilever tip

16. Alberts Molecular Biology of the Cell 4e © 2002 Garland Science Single-molecule mechanics of titin by AFM

17. Stepping of myosin V by high-speed AFM 37 nm Kodera, N. et al. Nature 468, 72 (2010)

18. Other important single-molecule techniques include: patch clamp super-resolution optical microscopies with active control of fluorophore emission fluorescence polarization magnetic tweezers imaging by cryoelectron microscopy, electron tomography