Combined High-Resolution Optical Tweezers and Multicolor Single-Molecule Fluorescence with an Automated Single-Molecule Assembly Line.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single-molecule assembly. The multicolor allows the simultaneous observation of multiple molecules or multiple degrees of freedom, which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, with a reliable alignment scheme, which will allow three independent fluorescent probe or FRET measurements and also increases flexibility in the choice of fluorescent molecules.

Combined Force Ramp and Equilibrium High-Resolution Investigations Reveal Multipath Heterogeneous Unfolding of Protein G.

Over the past two decades, one of the standard models of protein folding has been the "two-state" model, in which a protein only resides in the folded or fully unfolded states with a single pathway between them. Recent advances in spatial and temporal resolution of biophysical measurements have revealed "beyond-two-state" complexity in protein folding, even for small, single-domain proteins. In this work, we used high-resolution optical tweezers to investigate the folding/unfolding kinetics of the B1 domain of immunoglobulin-binding protein G (GB1), a well-studied model system.