Exploring the mechanome with optical tweezers and single molecule fluorescence

Abstract

The combination of optical tweezers and single molecule fluorescence into an instrument capable of making combined, coincident measurements adds an observable dimension that allows for the examination of the localized effects of applied forces on biological systems. This technological advance had remained elusive due to the accelerated photobleaching of fluorophores in the presence of the high photon flux of the optical trap. This problem was circumvented by alternately modulating the trapping and fluorescence excitation laser beams, a technique named IOFF. Results show that our solution extends the longevity of Cy3 fluorophores by a factor of 20 without compromising the stiffness of the optical trap. This versatile arrangement can be extended to other fluorophores and was applied to unzip a 15 base pair region of dsDNA and to induce reversible conformational changes in a dsDNA hairpin labeled with a FRET pair. Next, this work developed an immobilization strategy and two single molecule assays for the CIpX ATPase, an enzyme capable of unfolding substrates that have been targeted for proteolytic degradation. In the first assay, which employs single molecule fluorescence, CIpX was found to unfold and translocate pre-engaged GFP substrates with a time constant of 22 s at saturating ATP concentrations, a rate that is 8-fold faster than bulk measurements clouded by binding and unbinding events. The second assay measured the strength of the ClpX-substrate interaction with optical tweezers. Results show that CIpX holds on to its substrates with forces on the order of 55 pN regardless of the nature and concentration of the nucleotide in solution.

(cont.) Finally, optical tweezers were used to characterize the rheological properties of methylcellulose and polarized cells, to quantify the mechanical properties of bacteriophage, and to measure the forces generated by a cellular actin spring.