We have demonstrated a combination of single molecule force clamp spectroscopy and solvent substitution that captures the presence of solvent molecules in the transition state structure of a protein. We measure the effect of solvent substitution on the rate of unfolding of the I27 titin module, placed under a constant stretching force. From the force dependency of the unfolding rate, we can determine properties of the molecular architecture of the unfolding transition state structure. We find that water molecules play an active role in the unfolding transition state structure, bridging important beta-strands within the protein. Using force clamp spectroscopy we can probe this solvent bridging in more detail by replacing water with other solvents, such as the protecting osmolyte glycerol. Our results show that solvent composition is important for the mechanical function of proteins. Furthermore, given that solvent composition is actively regulated in vivo, it may represent an important modulatory pathway for the regulation of tissue elasticity and other important functions in cellular mechanics.
Research Topics
Our Atomic force microscope manipulations of single polysaccharide molecules have expanded conformational chemistry to include force-driven transitions between the chair and boat conformers of the pyranose ring structure. We postulate that the conformational changes result from the torque generated by the glycosidic bonds when a force is applied to the polysaccharide molecule. Glycosidic bonds act as mechanical levers, driving the conformational transitions of the pyranose ring. When the glycosidic bonds are equatorial (e), the torque is zero, causing no conformational change. However, when the glycosidic bond is axial (a), torque is generated, causing a rotation around COC bonds and a conformational change. We have investigated the conformations of a variety of polysaccharide molecules such as amylose, cellulose, pectin, etc, with different types of linkages and largely confirmed the torque hypothesis. Furthermore our observations are well supported by ab-initio calculations that show that readily reproduce the observed extensibility of the polysaccharide molecules upon undergoing conformational changes. The torque hypothesis readily predicts the number of transitions observed in pyranose monomers with 1a-4a linkages (two), 1a-4e (one), and 1e-4e (none). Our results demonstrate single-molecule mechanochemistry with the capability of resolving complex conformational transitions in the Ångstrom scale.
Thioredoxins are enzymes that catalyze disulfide bond reduction in all living organisms. While catalysis is thought to proceed through a substitution nucleophilic bimolecular (SN2) reaction, the role of the enzyme in modulating this chemical reaction is unknown. Here we use single molecule force-clamp spectroscopy to probe the catalytic mechanism of E. coli thioredoxin (Trx). We apply mechanical force in the range of 25-450 pN to a disulfide bond substrate and monitor the reduction of these bonds by individual enzymes. Our results suggest that substrate conformational changes may be important in the regulation of Trx activity under conditions of oxidative stress and mechanical injury, such as those experienced in cardiovascular disease. Furthermore, single molecule atomic force microscopy (AFM) techniques, as shown here, can probe dynamic rearrangements within an enzyme's active site which cannot be resolved with any other current structural biological technique.
The mechanism by which mechanical forces regulate the kinetics of a chemical reaction is unknown. We use single molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds via the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is of common occurrence in mechanically stressed proteins. Our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property which has never been explored by traditional biochemistry. Furthermore, our work also suggests that the kinetics of any chemical reaction that results in bond lengthening will be force dependent.
The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure dependent elasticity. We use protein engineering and single molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behavior of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.
Force-clamp spectroscopy is a novel platform to study protein folding. The coordinate for the unfolding reaction is known (end-to-end length), the unfolded state is well defined and can be controlled over wide ranges, and the folding trajectory can be followed in a single protein over time. We use two force-clamp protocols: force-quench and force-ramp. In contrast to the traditional two-state folding reactions observed in solution biochemistry, our folding trajectories from highly extended unfolded states are continuous and marked by several distinct stages. The time taken to fold is exponentially dependent on the stretching force applied during folding. While chain entropy makes a small contribution to the collapse, we have found that most of the driving force is hydrophobic and varying widely depending on the dihedral space traversed by the folding trajectory. This collapse mechanism is common to highly extended proteins, including non-folding elastomeric proteins like PEVK from titin.
One big advantage of our approach is that denaturing forces and extension can be controlled not only in magnitude but also in their direction. For example, we discovered that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems. Another surprising finding was the discovery that ankyrin, a protein made of multiple repeats, upon pulling, unfolds in a piecewise manner. The piecewise unfolding of multiple ANK repeats could behave like multiple buffers linked in series; to resist damagingly high forces, ANK repeats can be sacrificed and extended one at a time, without the whole protein losing its tertiary structure. Both of these discoveries are completely novel and could not have been anticipated from solution biochemistry.
