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Review
. 2020 May 14;5(20):11271-11278.
doi: 10.1021/acsomega.0c00814. eCollection 2020 May 26.

Force Spectroscopy on Single Molecules of Life

Soumit S Mandal  1
Affiliations
Review

Force Spectroscopy on Single Molecules of Life

Soumit S Mandal. ACS Omega. .

Abstract

Biomolecules such as nucleic acids and proteins constitute the cells and its organelles that form the crucial components in all living organisms. They are associated with a variety of cellular processes during which they undergo conformational orientations. The structural rearrangements resulting from protein-protein, protein-DNA, and protein-drug interactions vary in spatial and temporal length scales. Force is one of the important key factors which regulate these interactions. The magnitude of the force can vary from sub-piconewtons to several thousands of piconewtons. Single-molecule force spectroscopy acts as a powerful tool which is capable of investigating mechanical stability and conformational rearrangements arising in biomolecules due to the above interactions. Real-time observation of conformational dynamics including access to rare or transient states and the estimation of mean dwell times using these tools aids in the kinetic analysis of these interactions. In this review, we highlight the capabilities of common force spectroscopy techniques such as optical tweezers, magnetic tweezers, and atomic force microscopy with case studies on emerging applications.

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Conflict of interest statement

The author declares no competing financial interest.

Figures

Figure 1
Figure 1
Schematics representing optical tweezers (OT)-based assays. (A) The assay involves bringing a trapped silica bead coated with kinesin motor molecules (green) to the microtubule attached to the surface of a trapping chamber. The force and displacement generated by kinesin as it traverses along the microtubule are determined from the displacement of the bead in the optical trap. (B) One end of a nucleic acid tether is attached to a silica bead trapped at the focus of an infrared laser, while the other end is attached to another similarly optically trapped bead to generate a so-called dumbbell assay. The other bead can also be held in a micropipette tip. (C) OT assay to study the unfolding/refolding of a protein. The intertrap distance d and X1, X2 represent the deflection of each bead out of their respective trap center. With R being the bead’s radius, the extension of a stretched tether comprising DNA handles and protein is Xtether = d – 2R – X1 – X2. With calibrated trap stiffnesses k1 and k2, the force acting on the system is F = k1X1 = k2X2 = keff (X1 + X2), where keff = (1/k1 + 1/k2)−1 is the effective spring constant. (D) Experimental scheme of investigating protein droplet fusion. One laser beam is used to hold one protein droplet at a fixed position, while another protein droplet, trapped by a second laser, is moved toward the first droplet at a constant velocity. As these droplets are brought into close proximity, protein droplets coalesced rapidly (adapted and modified from ref (19)). (E) Representative constant-velocity trajectory showing a two-state unfolding/refolding event. (F) Extension–time trajectories at three constant mean forces showing a molecule fluctuating between two states.
Figure 2
Figure 2
(A) In magnetic tweezers (MTs), a magnetic field acts in a direction perpendicular to the DNA axis, which limits the angular rotations of the bead while exerting an upward pulling force. Rotating the magnets will introduce a torque. When torque buildup is greater than the bucking torque, coiled structures form that lead to the bead being pulled toward the substrate. Topoisomerase action relaxes the supercoil in the DNA, leading to changes in the bead height, with ΔZ being proportional to the number of supercoils removed. (B) Representative hat curves for a bead with an attached DNA. At low force, the hat curves are symmetric. At higher forces, untwisting causes DNA melting or the formation of Z-DNA, whereas overtwisting induces the formation of P-DNA, which suppresses the formation of supercoils and the resulting DNA contraction.
Figure 3
Figure 3
(A) Schematic representing an atomic force microscope. A DNA molecule is attached to the substrate on the piezoelectric stage and the cantilever tip. As the piezoelectric stage is retracted along the axial direction, the separation between the cantilever and the sample surface increases. The cantilever deflection generates a force acting on the DNA. The extension of DNA is calculated from the distance between the AFM tip and the substrate. (B) AFM measurements of a polyprotein construct. The movement of the piezoelectric positioner is represented by ΔZp. Initially the protein is in a relaxed state. Stretching this protein to near its folded contour length, L1, requires a force that is measured as a deflection of the cantilever. Stretching further increases the applied force, which triggers the unfolding of a domain, increasing the contour length of the protein and relaxing the cantilever back to its resting position. Further stretching removes the slack and brings the protein to its new contour length L2 (adapted and modified from ref (23)). (C) Characteristic force–extension sawtooth pattern curve resulting from stretching a polyprotein. Each sawtooth peak corresponds to the unfolding of one of the domains, while the last peak arises from the detachment of the molecule from the substrate or AFM tip. The amplitude of the sawtooth unfolding force peak measures the force at which the protein domain unfolds. Dotted lines correspond to fits of the worm-like chain model of polymer elasticity to the experimental data. The contour length increment, ΔLc, measures the length increment upon protein unfolding.
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