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Exploring Reaction Pathways of Single-Molecule Interactions through the Manipulation and Tracking of a Potential-Confined Microsphere in Three Dimensions

Published online by Cambridge University Press:  01 February 2011

Wesley P. Wong ,
Volkmar Heinrich  and
Evan Evans
Wesley P. Wong
Affiliation:
Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA. Department of Physics, Harvard University, Cambridge, MA 02138, USA.
Volkmar Heinrich
Affiliation:
Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
Evan Evans
Affiliation:
Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA. Department of Physics, Boston University, Boston, MA 02215, USA.
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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.

Type
Research Article
Information
MRS Online Proceedings Library (OPL) , Volume 790: Symposium P – Dynamics in Small Confining Systems–2003 , 2003 , P5.1
Copyright
Copyright © Materials Research Society 2004

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