Biological Chemistry and Molecular Pharmacology
We are interested in understanding the physical basis of how biological systems work at the nanoscale, with a focus on the role of mechanical force. More specifically, we investigate how force regulates the structure and dynamics of interactions between and within single-molecules, and how this in turn can affect the functioning and malfunctioning of biological systems. To accomplish this, my group develops and applies novel tools in single-molecule manipulation and detection, combining approaches from a variety of disciplines, including physics, molecular biology, chemistry, and engineering. Some key research projects that we are working on are highlighted below.
1. Novel high-throughput methods in single-molecule manipulation, including single-molecule centrifugation
The precise manipulation and observation of single molecules provides a detailed view of biomolecular systems that is unobscured by ensemble averaging, and that reveals non-equilibrium and force-dependent behavior. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. My group is developing a new approach to solve these issues: massively parallel single-molecule force measurements using centrifugal force. Our new instrument, the Centrifuge Force Microscope (CFM), can perform thousands of single-molecule force experiments in parallel, enabling measurements to be made in minutes rather than in days.
Drawing from techniques ranging from optical tweezers and single-molecule fluorescence to modern methods in molecular biology and surface/linkage chemistry, my group develops novel approaches as needed to answer interesting biological and biophysical questions.
2. Force-regulated molecular interactions in the circulatory system, including studies on the vascular protein von Willebrand factor
Molecules in the circulatory system operate in a dynamic environment of forces and flow. This is particularly true for vascular molecules involved in cell adhesion. Single-molecule force studies give us a way to understand how these molecular systems work, by serving as both an analogue for forces experienced in the body, and as a probe for exploring the interaction energy landscape.
We have been studying the vascular protein von Willebrand Factor (vWF), which is responsible for tethering platelets both to each other and to sites of trauma under conditions of high shear. For example, in collaboration with Timothy A. Springer’s group we have shown that the A2 domain of vWF functions as a force-activated single-molecule switch in a key molecular feedback loop that regulates primary hemostasis. Specifically, by characterizing the kinetics of this system using single-molecule force spectroscopy, we have shown that hydrodynamic forces in the circulation can act as a “cofactor” for enzymatic cleavage of A2 by the ADAMTS13 enzyme. We are continuing to investigate the dynamics of molecular transitions within vWF (e.g. bond formation and rupture, unfolding/refolding transitions, and enzymatic cleavage) under conditions of externally applied tension and hydrodynamic flow, which should lead to new insights into von Willebrand disease, the most common hereditary bleeding disorder.
3. Other studies on the role of mechanical force in nanoscale biology, include:
a) Structure and dynamics of bacterial chromosomesb) Development of force-based nanoscale molecular therapeuticsc) Dynamics of Actin-associated proteinsd) Development of quantitative models and methods of analysis for biomolecular interactions
We are interested in understanding the physical basis of how biological systems work at the nanoscale, with a focus on the role of mechanical force.
K. Halvorsen, W.P. Wong^, “Massively parallel single-molecule manipulation using centrifugal force,” Biophysical Journal – Letters 98 (11), L53-L55 (2010).
X. Zhang*, K. Halvorsen*, C.-Z. Zhang, W.P. Wong^, and T.A. Springer^, “Mechanoenzymatic cleavage of the ultralarge vascular protein, von Willebrand Factor,” Science 324 (5932), 1330-1334 (2009).
E. Evans, K. Halvorsen, K. Kinoshita, and W.P. Wong, “A new approach to analysis of single molecule force measurements,” in Handbook of Single Molecule Biophysics, P. Hinterdorfer, ed., Springer (2009).
W.P. Wong*^ and K. Halvorsen*, "Beyond the frame rate: Measuring high-frequency fluctuations with light intensity modulation," Optics Letters 34 (3), 277-279 (2009).
*contributed equally^corresponding author