Exploring Reaction Pathways of Single-Molecule Interactions through the Manipulation and Tracking of a Potential-Confine
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Exploring Reaction Pathways of Single-Molecule Interactions through the Manipulation and Tracking of a Potential-Confined Microsphere in Three Dimensions Wesley P. Wong1,3, Volkmar Heinrich1, and Evan Evans1,2 1
Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA. Department of Physics, Boston University, Boston, MA 02215, USA. 3 Department of Physics, Harvard University, Cambridge, MA 02138, USA. 2
ABSTRACT 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 [3]) to encompass near-equilibrium forward/reverse transitions of weak-complex interactions with multiple binding configurations and more than one transition pathway. INTRODUCTION “Weak” biomolecular bonds are characterized by much smaller binding energies than covalent bonds, and typically are on the order of 10-30 kBT in thermal energy units (here kB is the Boltzmann constant, and T ~300 K is temperature, i.e. kBT ≈ 4×10-21 J). Therefore, in the aqueous environment of the cell, thermal activation alone is capable of disrupting a weak bond, which leads to characteristic bond lifetimes ranging from microseconds to
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