Optical tweezers for protein folding studies

Optical tweezers were invented by Arthur Ashkin, who was honored for this achievement with The Nobel Prize in Physics 2018. For most biological applications, micron-sized glass or plastic beads are used as force probes. These beads are attracted to the center of a tightly focused laser beam – they are “optically trapped”. While the complete description of how the light interacts with the bead is beyond the scope of this page, an intuitive understanding of optical trapping can be gained using ray optics considerations. If the bead is displaced from the center of the beam, asymmetric refraction causes a deflection of the laser beam after passing through the bead. The photons therefore experience a change of momentum, i.e. a force. Conservation of momentum then requires that the bead experiences an equal force that attracts it to the center of the laser beam. Optical tweezers can therefore be used to exert force on single macromolecules that are tethered to the bead, and from the deflection of the beam, the force on the molecule can be precisely measured.

optical tweezers: ray optics scheme

Optical tweezers operate in the piconewton force range. Many mechanical processes in the cell fall into the same range. Force applied to a folded protein acts as a denaturant. However, in contrast to its bulk counterparts like urea or extreme pH, force acts locally. The selective application of denaturing conditions makes it possible to probe individual parts or complex systems. For instance, we can selectively destabilize a ribosome-bound nascent protein to study its folding without denaturing the ribosome itself. Similarly, molecules in solution, such as molecular chaperones, are unaffected by denaturing conditions applied to a client polypeptide. In addition, the single-molecule nature of the measurement circumvents the problem of protein aggregation that often hampers folding studies of structurally complex proteins. Single-molecule force spectroscopy with optical tweezers is therefore an ideal tool for studying the folding of protein on the ribosome and the mechanism of action of molecular chaperones.

ribosome-nascent chain complex pulled with optical tweezers

Spectroscopic techniques, such as circular dichroism or tryptophan fluorescence spectroscopy, have been instrumental for determining protein folding mechanism. One drawback of these “classic” approaches is that they record average properties of large ensembles (typically millions to billions of copies) of a given protein. Important, optical tweezers (and other single-molecule) experiments avoid “ensemble averaging”. This aspect can be important, because average properties can be misleading. Imagine that you roll a die many times. The number of pips averaged over all events is 3.5, but there is no face of the die with 3.5 pips, illustrating that an ensemble average may not reflect any state that a system can actually adopt. By studying folding one molecule at a time, complex folding pathways can be resolved.

illustration of ensemble averaging - roll of a die