1. Shaobin Guo 11/20/2012

2. various types of Single-molecule force spectroscopy Optical tweezers Magnetic tweezers Atomic force microscopy (AFM) Micro-needle manipulation Biomembrane force probe Flow-induced stretching

3. Single-molecule manipulation capacity Length: 10 -10 - 10 -4 m / measurement of RNA polymerase advancing a single base pair to manipulation of cells Force: 10 -14 - 10 -8 Newton / assaying nucleic acid folding kinetics to mechanical disruption of covalent bonds

4. Optical tweezers (Trap) Lasers: near-infrared wavelengths (800-1100 nm) High numerical aperture (NA) microscope objective (at least 1.2) An approximate linear spring for small displacements (~150 nm) Position detection: back-focal plane (BFP) interferometry Calibration: position detector and trap stiffness

5. applications Interaction assay Tethered assay Infrared laser silica bead kinesin molecule microtubule RNA polymerase molecule Dumbbell assay

6. Limitations and Drawbacks Require optically homogeneous preparations and highly purified samples for high resolution trapping. Lack selectivity and exclusivity Local heating Optical damage Limited range of applied force (0.1-100 pN) and range of displacement (~150 nm or less)

7. magnetic tweezers A pair of permanent magnets Inverted microscope with a charge-coupled device (CCD) Able to rotate super-paramagnetic beads ranging from 0.5 to 5 um Force-clamp property: an effective stiffness on the order of 10 -6 pN/nm Free from sample heating, photodamage and other problems related to optical tweezers Drawbacks: not versatile, unable to directly measure rotational torque generation, limited to slow and large displacements

8. Electromagnetic tweezers Sharp electromagnetic tips Able to exert forces in excess of 1 nN Easy to control force and rotation by changing current Three-dimensional position control Drawbacks: cumbersome feedback control, sophisticated pole pieces, lack of sensitivity and heating.

9. Atomic Force microscopy (AFM) A very high-resolution type of scanning probe microscopy, with demonstrated sub- nm resolutionscanning probe microscopy Able to measure inter- and intramolecular interaction forces with pN resolution Simple and rapid sample preparation, and the ability to conduct measurements under near-physiological conditions

10. applications Study of molecular bond’s rupture: covalent bonds to protein unfolding Force-clamp spectroscopy by AFM Investigation of supramolecular assemblies Combination of AFM imaging with force mapping and spectroscopy to image the surface topology at high resolution and measure the unbinding or unfolding forces at well-defined locations. High-speed AFM which makes it possible to follow molecular events in real time.

11. Limitations and Drawbacks Narrow useful force range resulting from large size and relative high stiffness of the cantilevers Lack of specificity

13. Background Regiospecific thiol-disulfide exchange reactions: electrons are reshuffled between a thiolate and a disulfide bond via an S N 2 mechanism that produces a different disulfide and a new thiolate

14. problems with bulk techniques Inability to differentiate disulfide isomers Variations among different techniques used to analyze reactions and subsequent divergent interpretations Interference from the reverse reaction

15. experiment set-up Cantileve r Tip Piezoelectric scanner Force: 250 pN

16. Reduction events from polypeptides containing single disulfide bond 14.5 nm 10 nm (I27 32– 75 ) 8 (I27 24– 55 ) 8 Predicted extension after the reduction of a disulfide bond

17. Reduction events from polypeptides containing two disulfide bonds (I27 2S-S ) 4

18. Cleavage of disulfide 32-75 in I27 2S-S

19. Pathways available for the reduction of disulfide 24–55 after the reduction of disulfide 32–75

21. At high concentrations of L-Cys the frequency of the 10 nm steps increases at the expense of the 3 and 8nm steps

23. Thanks! Questions?