Protein-Folding Studies using Hybrid TIRF SmFRET-Magnetic Tweezers

Optical tweezers are a useful research tool for applying forces to single proteins and measuring the resulting end-to-end extension increases that accompany unfolding and other force-induced conformational changes. They do, however, have a few limitations: they can only observe changes in extension along the axis of force application; the accompanying changes in extension must be on the order of at least a few nanometers to be resolved above the noise of the instrument; and particularly for multi-domain and polyproteins, an observed change in extension cannot always be localized to a specific region of sequence. Our experimental setup overcomes these limitations by measuring distance changes using single-molecule Forster resonance energy transfer (smFRET) produced from a total internal reflection fluorescence (TIRF) microscope incorporating magnetic tweezers. Individual protein molecules are conjugated to FRET-paired dyes and functionalized DNA handles using disulfide and click chemistry. These handles tether each molecule to the surface of a coverslip on one end and to a paramagnetic bead on the other. By simultaneously measuring donor and acceptor intensities, as well as magnet position, we are able to monitor force-induced conformational changes in multiple protein systems.