Optical tweezers -

measuring forces in the microscopic world

 

Our laboratory specializes in optical tweezers, a single molecule mechanical manipulation technique that allow us to track the activity of individual enzymes, measure the force required to unfold a single protein molecule, or determine strength of binding between individual protein molecules.

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In optical tweezers, a single protein molecule is tethered between two beads using DNA as molecular handles. One bead is in the optical trap and the other bead is held by suction on the tip of a micropipette. The target protein is mechanically unfolded and refolded by moving the bead in the optical trap away or towards the bead on the micropipette, thus stretching or relaxing the tether tension between the two beads.

For example, the mechanical unfolding trajectory shown below for the regulatory domain of PKA reveals three unfolding transitions. We mapped each unfolding transition to the corresponding protein structure to determine which secondary and tertiary structural elements unravel in each unfolding transition.

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To provide an atomistic description of the mechanical unfolding pathway seen in our optical trap measurements, we perform steered molecular dynamic simulations of the protein in collaboration with Prof. Emanuele Paci from the University of Leeds. Below are two simulations of the mechanical unfolding of the PKA regulatory subunit in the presence and absence of cAMP.

 

How do optical tweezers work?

Optical tweezers focus a laser beam through a lens to form a “trap”. The interaction of small dielectric objects, such as proteins, with a focused Gaussian beam generates a force in the direction of the field gradient drawing it toward the center of the beam. When the object is displaced away from the center, a restoring force arises.

optical-trapThe restoring force can be measured directly by projecting the beam onto a position-sensitive detector and measuring both its intensity and deflection. Because this restoring force is proportional to the stiffness of the trap (k) and to the displacement of the object from the center of the trap (Δx) , the force required for displacement of the object can also be determined from this displacement using Hooke’s law: F = kΔx.