Stretching of small peptide molecules:
This work was originally established in collaboration with Dr Jun Miyake at the former National Institute for Bioscience and Human-Technology, Japan. Below-resonance MAD mode atomic force microscopy was used to obtain a continuous measurement of stiffness whilst stretching single peptide molecules in the alpha-helical state. To isolate single molecules between tip and sample we utilized the sulphur-gold bond. By having a very small percentage of active peptides with a cysteine available for attachment to the gold coated tip it was possible to increase the chance of single molecule stretching events. Preliminary results indicated a consistent value for hydrogen bonding within the alpha helix.
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M. A. Lantz, S. P. Jarvis, H. Tokumoto, T. Martynski, T. Kusumi, C. Nakamura and J. Miyake "Stretching the a-helix - A direct measure of the hydrogen bond energy of a single peptide molecule", Chem. Phys. Lett., 315, 61 (1999).
M. Kageshima, M. A. Lantz, S. P. Jarvis, H. Tokumoto, S. Takeda, A. Ptak, C. Nakamura, J. Miyake Insight into conformational changes of a single A-helix peptide molecule through stiffness measurements, Chem. Phys. Lett., 343, 77-82 (2001).
M. Kageshima, S. Takeda, Arkadiusz, A. Ptak, C. Nakamura, S.P. Jarvis, H. Tokumoto and J. Miyake, “Measurement of Intramolecular Energy Dissipation and Stiffness of a Single Peptide Molecule by Magnetically Modulated Atomic Force Microscopy”, Japanese Journal of Applied Physics, in press |
Single molecule ligand-receptor interactions:
The high affinity for biotin-avidin association and stability of the subsequent complex has made the biotin-avidin protein a principle component for binding assay techniques and recently a model system for investigating ligand-receptor interactions using AFM. This work represents the first application of FM-AFM to investigate the dynamics of biotin-avidin unbinding. To operate effectively in a liquid environment, the frequency modulation is implemented magnetically. AFM tips are covalently functionalised with biotin using a poly(ethylene glycol) cross-linker and avidin is electrostatically immobilised on a mica surface. |
The figure to the left shows the dynamics of the biotin-avidin interaction during an AFM measurement. In (A), an oscillating cantilever approaches the surface to form the biotin-avidin bond. In (B), the tip is retracted to apply an unloading force to the biotin-avidin complex. Lastly in (C), the complex unbinds.
During the measurement, the frequency shift of the cantilever is recorded and converted to a force. The frequency shift curves have shown an adhesion peak that represents the stretching of the
PEG linker, subsequent breaking of the bond and further novel rebinding effects. We have measured forces up to 300pN due to the higher loading rate, which
corresponds to the resonance frequency (approx. 20 kHz) of the AFM cantilever. Thus, the use of higher loading rates allows for the possibility to explore undiscovered energy barriers in bond-dissociation pathways. |
Michael J. Higgins, Christian K. Riener, Takayuki Uchihashi, J.E. Sader, Rachel McKendry and Suzanne P. Jarvis, “Frequency modulation atomic force microscopy: a dynamic measurement technique for biological systems”, Nanotechnology 16 (2005) S85-S89.
M.J. Higgins, J.E. Sader, and S.P. Jarvis, “Frequency modulation AFM reveals individual intermediates associated with each unfolded I27 titin domain,” Biophysical Journal 90, 640-647 (2006). |
