Force spectroscopy measurements of non-covalent interactions on the supramolecular and single molecule levels

Non-Covalent interactions are ubiquitous and play a vital role in all biological and chemical systems. Atomic force spectroscope (AFM) based approaches have been applied to study the non-covalent interactions and protein aggregates stabilized by non-covalent interactions at two nano scale levels: the supramolecular level and the single molecule level.;At the supramolecular level, the AFM nanoindentation approach for the first time has been employed to study the mechanical properties of insulin amyloid fibrils and insulin crystals. Our results show a small elasticity variation along the fibril axis indicating that a relatively uniform structure is kept within each individual insulin amyloid fibril. However a significant elasticity difference among fibrils exists, suggesting that there are heterogeneous structures among fibrils though they are synthesized under the same conditions. Moreover, the measured modulus of insulin crystals falls between the elastic modulus values of insulin amyloid fibrils measured in the direction of the fibril axis and the direction perpendicular to the fibril axis, implying that molecule packing within insulin amyloid fibrils is very anisotropic.;In order to improve the measurement accuracy, methodological development of nanoindentation has been conducted, including the use of a new data reduction approach that increases the accuracy and precision of the elasticity measurements by significantly reducing the uncertainty in determining the contact point between the AFM probe and the sample and in the finite thickness correction to eliminate the substrate effect on the sample's elasticity. This approach can be easily adopted to study the mechanical properties of other samples. As a detailed example, the study of mechanical properties of human retinal pigment epithelium melanosomes has been described.;At the single molecule level, the AFM based force spectroscopy has been applied to study the specific interactions between ligand (biotin) and receptor (streptavidin). The results from previous measurements of this interaction are inconclusive and require methodological improvements to accurately extract the kinetic parameters. The two bond rupture model has been developed and applied to the data analysis, which successfully explains the measured broad distribution of rupture forces. The kinetic parameters extracted from our measurements are consistent with the energy landscape predicted by molecular dynamics simulations. In addition, this model also predicts that if the presence of multiple bonds ruptures in force spectroscopy data is ignored, the noise-limited detection of rupture forces might lead to an incorrect interpretation of shape of the potential of mean force. In particular, the standard data analysis procedures might lead to the presence of artificial internal barriers. Application of the proposed force spectroscopy model facilitates an accurate interpretation of experiments.;Finally, to quantitatively characterize bimolecular reactions of association and dissociation at a single molecule level, a new approach to study kinetics of the association part of a reaction is proposed. This approach is based on the statistical analysis of the binding probability between single molecules in force spectroscopy measurements. The activation energy of biotin-streptavidin binding that is extracted from our measurements shows a reasonable magnitude compared with the activation energy of biotin-streptavidin dissociation. The developed model allows us to obtain the complete kinetic information of an interaction from one set of force spectroscopy measurements and can be extended to other single molecule force spectroscopy measurements.