Optical Tweezers Approaches for Probing Multiscale Protein Mechanics and Assembly

Abstract

Multi-step assembly of individual protein building blocks is key to the formation of essential higher-order structures inside and outside of cells. Optical tweezers is a technique well suited to investigate the mechanics and dynamics of these structures at a variety of size scales. In this mini-review, we highlight experiments that have used optical tweezers to investigate protein assembly and mechanics, with a focus on the extracellular matrix protein collagen. These examples demonstrate how optical tweezers can be used to study mechanics across length scales, ranging from the single-molecule level to fibrils to protein networks. We discuss challenges in experimental design and interpretation, opportunities for integration with other experimental modalities, and applications of optical tweezers to current questions in protein mechanics and assembly.

Keywords: collagen; fibrillar proteins; microrheology; optical tweezers (OT); protein assemblies; protein mechanics; protein structure/folding; single molecule.

FIGURE 1

Principles of single-molecule force spectroscopy (SMFS) with optical tweezers (OT). (A) Schematic of an optical tweezers stretching experiment with collagen. The displacement of the bead center from the trap center Δx and the trap stiffness κ provide the force applied to stretch the molecule. A Worm-Like Chain (WLC) model (red curve) can be used to fit the resulting force extension curve (black dots). Adapted from Wieczorek et al. (2015) with permission. (B) Experimental scheme for OT-SMFS experiments with short proteins. Prion proteins (PrP) are tethered to polystyrene beads via DNA handles (left). The PrP unfolds and refolds to its native state, dependent on the applied force, as a two-state system (middle). PrP dimers linked at their termini lead to complex force extension curves with multiple intermediates, and more remarkably, adopt a misfolded dimer structure at low force rather than two independently folded domains (right). Adapted from Figure 2 in Dee and Woodside (2016) with permission. (C) Illustration of measurements of the bending modulus of a collagen fibril. The inset illustrates the highly ordered lateral organization of collagen molecules within a single fibril, which creates a characteristic “D-banding” pattern (dark/light stripes). The optically trapped bead is used to apply bending deformations to a fibril (left). The resulting force-displacement curve reveals the force required for different applied lateral bending strains (right). Adapted from Dutov et al. (2016) with permission under CC BY license.

FIGURE 2

Optical-tweezers based microrheology (OT-MR) measurements of protein network formation and remodeling. (A) Analysis of trapped bead motion is used to determine how the microscale viscoelastic environment changes during assembly and growth of collagen fibrillar networks (schematic upper panel). Arrows indicate thermally driven fluctuations of the particles within the optical traps, used in passive MR experiments to determine the complex shear modulus of the surroundings. For collagen, it was found that elastic moduli and their spread tend to increase during assembly (lower panel, circles), as the local environment becomes more heterogeneous. The red line and shaded region indicate the range of optical trap elastic moduli G′_trap measured for this bead size, while the red dots at zero time indicate the elastic moduli of trap + collagen solutions in acidic conditions, where assembly cannot occur. Filled black circles indicate repeated measurements on the same bead at multiple times during assembly, illustrating distinct evolutions of local mechanics. These experiments found G′_max (at fixed frequencies) to increase with the same sigmoidal kinetics as the optical turbidity used to measure growth of the network (blue curve). Adapted from (Shayegan and Forde, 2013) with permission under CC BY license. (B) OT-MR probes the effect of transient protein-protein interactions that catalyze protein network assembly. Collagen assembly is accelerated by telopeptides, short regions flanking the triple helix (shown as small forked ends in the schematics). Even in acidic conditions where assembly cannot occur, solutions of collagens with intact telopeptides (red dashed line) exhibit a significantly greater G’ at low frequencies than collagens with telopeptides enzymatically removed (purple dashed line). The decrease in G’ can be detected in real time, as enzymes gradually remove telopeptides (colored markers), thereby reducing protein-protein interactions. Adapted from Shayegan et al. (2016) with permission from Elsevier. (C) It may also be possible to extract the kinetics of transient crosslinking proteins with OT-MR, as found in bulk rheology experiments on actin. At shorter times/higher frequencies (right inset), only short-range bending fluctuations of the actin filaments can occur, while at longer times/lower frequencies (left inset) actin filaments can undergo larger-scale deformation enabled by unbinding of a crosslinking protein (red circle). Thus, G″ of a crosslinked actin gel exhibits a local maximum at a frequency corresponding to the unbinding rate (inversely proportional to the characteristic unbinding time t_off) of the protein crosslinker. Adapted from Broedersz et al. (2010) with permission (copyright 2010 by the American Physical Society).