Weak non-covalent interactions between single molecules govern many aspects of microscopic biological structure and function, e.g. cell adhesion, protein folding, molecular motors and mechanical enzymes. The dynamics of a weak biomolecular bond are suitably characterized by the kinetic transport of molecular states over an effective energy landscape defined along one or more optimal reaction pathways. Motivated by earlier developments [1,2], we present a novel method to quantify subtle features of weak chemical transitions by analyzing the 3D Brownian fluctuations of a functionalized microsphere held near a reactive substrate. A weak optical-trapping potential is used to confine motion of the bead to a nanoscale domain, and to apply a controlled bias field to the interaction. Stochastic interruptions in the monitored bead dynamics report formation and release of single molecular bonds. In addition, variations in the motion of a bead linked to the substrate via a biomolecule (a protein or nucleic acid) signal conformational changes in the molecule, such as the folding/unfolding of protein domains or the unzipping of DNA. Thus, energy landscapes of complex biomolecular interactions are mapped by identifying distinct fluctuation regimes in the 3D motion of a test microsphere, and by quantifying the rates of transition between these regimes as mediated by the applied confining potential.
The 3D motion of the bead is tracked using a reflection interference technique combined with high-speed video microscopy. The position of the bead is measured over 100 times per second with a lateral resolution of ∼3–5 nm and a vertical resolution of ∼1–2 nm. Crucial to the interpretation of results, a Brownian Dynamics simulation has been developed to relate the statistics of bead displacements to molecular-scale kinetics of chemical interactions and structural transitions. The experimental approach is designed to enlarge the scope of current techniques (e.g. dynamic force spectroscopy ) to encompass near-equilibrium forward/reverse transitions of weak-complex interactions with multiple binding configurations and more than one transition pathway.