Molecular Mechanics

In this work, we are focussed on molecular mechanics using micro-sensor technology. We have developed the lateral molecular-force microscope (LMFM) with a patented detection system and this technique is already demonstrating to be a unique tool for measuring and manipulating bio-molecular systems with nanometer resolution and femtoNewton sensitivity. Reducing the sensor size to the nanoscale, as pioneered in Bristol, will disclose an unprecedented level of detail of bio-molecular structures and their dynamic behaviour in vivo.

ultra soft cantilever

Comparison of ultra-soft cantilever with commercial contact mode AFM cantilevers (MLCT from Veeco, Santa Barbara, CA, USA) (scale bar is 200 mm).

The next strategic objective is to combine mechanical measurements of bio-molecules with other physical properties, in particular: electron transfer, polarisability and optical spectroscopy. The technology to access these combined measurements is not currently available, and we are in a unique position to integrate these new functionalities in our current nanosensors. We will develop, in an initial proof-of-concept stage, the methods and the procedures necessary to produce and use force sensors with the ability to measure extremely small currents or capacitance in a liquid environment. The following stage will require high-level interdisciplinary collaborations to address crucial questions in bio-molecular science and explore commercialisation avenues of these nanodevices.

Experimental Set-Up

Schematic of the LMFM

Experimental set up of the LMFM

Below is a diagram of the LMFM detection system and of the cantilever tethered to an MT via a kinesin molecule (not to scale). An evanescent field is generated above the glass coverslip by focusing a laser beam (1) off-axis at the back focal plane of a high NA objective lens (2). The light scattered (3) by the cantilever tip and the exit beam (4) are collected by the tube lens (5) and projected by a further 100× objective lens (6) onto a four-sector photodetector (7) where an interference pattern is generated. The diagram also highlights the constraints in the experimental measurement, as described in the text.

The kinesin head domains are required to bind following a straight line on the MT (x direction) and the cargo position is also constrained to a parallel line, as the cantilever will not allow significant rotations along the longitudinal axis or translations in the y and z directions. The black arrows emphasize the allowed direction of movement of the different parts of the system. (Inset) A CCD camera (not shown) records a TIRF image of the microtubules and the tip of the cantilever scattering the evanescent field (field of view 50 × 50 μm2 ).

Measuring the movement of kinesin

processive stepping of kinesin

The processive stepping of a single kinesin molecule

Processive stepping of a single kinesin molecule. Time course of a single kinesin molecule as it moves along an MT in 8.0 ± 0.4 nm steps at 1 mM ATP concentration. The superimposed parallel lines are 8.0 nm apart. The molecule binds to the MT after 0.12 s and it reaches a distance of 110 ± 5 nm from the centre position, before stalling at a force of 4.0 ± 0.2 pN. (Inset) The last short step (arrow) is enlarged and confirms that the system can clearly measure events lasting ≈3 ms. Data were recorded at 64 kHz (grey curve) and interpolated using cubic spline interpolation (black curve). The inset shows raw data.

Atomic Force Microscopy of Mx Bacteria

AFM analysis of Mx bacteria

Atomic force microscopy analysis of Mx bacteria

The images below show atomic force microscopy analysis of Mx bacteria. (A) A sinusoidal sideways movement is imparted to the sample stage in order to gently push a single adsorbed bacterium (blue sphere; pale-blue outer layer represents surface adhesins) against the cantilever. The change in the sample stage position (Δx1) required to achieve contact is measured upon addition of either ligand (CEACAM1 or Fn) or buffer/control protein. The graphs on the right show representative data measured for the change in the contact point position before (in red) and after (in green) the addition of (B) control, (C) CEACAM1, and (D) Fn. The red and green lines represent the mean value of the red and green points, respectively.

Working in this area

The following people are involved in this research:

Latest publications

  • Harniman,R.L., Vicary J.A., Hoerber, J.K.H., Picco L.M., Miles M.J.M. and An- tognozzi M. Methods for imaging DNA in liquid with lateral molecular-force microscopy Nanotechnology (2012). in Press
  • Scholz, T. et al. Processive behaviour of kinesin observed using micro-fabricated cantilevers. Nanotechnology 22, 5707 (2011).
  • Ulcinas, A. et al. Shear Response of Nanoconfined Water on Muscovite Mica: Role of Cations. Langmuir 27, 10351(2011).
  • Agnew, C. et al. Correlation of in situ mechanosensitive responses of the Moraxella catarrhalis adhesin UspA1 with fibronectin and receptor CEACAM1 binding. P Natl Acad Sci Usa 108, 15174 (2011).
  • Amrute-Nayak, M., Antognozzi, M., Scholz, T., Kojima, H. and Brenner, B. Inor- ganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II. J. Biol. Chem. 283, 3773 (2008).
  • Antognozzi, M. et al. A new detection system for extremely small vertically mounted cantilevers. Nanotechnology 19, 384002 (2008).

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