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. We demonstrate the ability to simultaneously measure angstrom-scale changes in tether extension and fluorescence signals. Simultaneous tweezers and fluorescence measurement are particularly challenging because of fluorophore photobleaching, even more so if multiple fluorophores are to be measured. Therefore, (1) fluorescence excitation and detection is interlaced with time-shared dual optical traps. (2) We investigated the photostability of common fluorophores. The mean number of photons emitted before bleaching was unaffected by the trap laser and decreased only slightly with increasing excitation laser intensity. Surprisingly, we found that Cy5 outperforms other commonly used fluorophores by more than fivefold. (3) We devised computer-controlled automation, which conserves fluorophore lifetime by quickly detecting fluorophore-labeled molecule binding, turning off lasers, and moving to add the next fluorophore-labeled component. The single-molecule assembly line enables the precise assembly of multimolecule complexes while preserving fluorophores.

Single molecule force spectroscopy at high data acquisition: A Bayesian nonparametric analysis.

Bayesian nonparametrics (BNPs) are poised to have a deep impact in the analysis of single molecule data as they provide posterior probabilities over entire models consistent with the supplied data, not just model parameters of one preferred model. Thus they provide an elegant and rigorous solution to the difficult problem encountered when selecting an appropriate candidate model. Nevertheless, BNPs' flexibility to learn models and their associated parameters from experimental data is a double-edged sword. Most importantly, BNPs are prone to increasing the complexity of the estimated models due to artifactual features present in time traces. Thus, because of experimental challenges unique to single molecule methods, naive application of available BNP tools is not possible. Here we consider traces with time correlations and, as a specific example, we deal with force spectroscopy traces collected at high acquisition rates. While high acquisition rates are required in order to capture dwells in short-lived molecular states, in this setup, a slow response of the optical trap instrumentation (i.e., trapped beads, ambient fluid, and tethering handles) distorts the molecular signals introducing time correlations into the data that may be misinterpreted as true states by naive BNPs. Our adaptation of BNP tools explicitly takes into consideration these response dynamics, in addition to drift and noise, and makes unsupervised time series analysis of correlated single molecule force spectroscopy measurements possible, even at acquisition rates similar to or below the trap's response times.

Randomizing phase to remove acousto-optic device wiggle errors for high-resolution optical tweezers.

Acousto-optic (AO) devices have been used extensively in optical tweezers because of their flexibility and speed; however, these devices have trap positioning inaccuracies that limit their usefulness, especially for high-resolution applications. We show that these inaccuracies are due to interference patterns within the AO device sound fields. We have devised a method that removes these inaccuracies by reducing the coherence of the sound fields by directly controlling and randomizing the phase of the radio frequency voltage input signal. We demonstrate that the trapping inaccuracies are eliminated, for both constant trap position and force-ramp measurements, and that no additional noise is added. We show that this random phase method is applicable to both acousto-optic modulator and deflector type devices and can be easily integrated via software upgrade into existing instruments.

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. Experiments were performed for GB1 both in and out of equilibrium using force spectroscopy. When the force was gradually ramped, simple single-peak folding force distributions were observed, while multiple rupture peaks were seen in the unfolding force distributions, consistent with multiple force-dependent parallel unfolding pathways. Force-dependent folding and unfolding rate constants were directly determined by both force-jump and fixed-trap measurements. Monte Carlo modeling using these rate constants was in good agreement with the force ramp data. The unfolding rate constants exhibited two different behaviors at low vs high force. At high force, the unfolding rate constant increased with increasing force, as previously reported by high force, high pulling speed force ramp measurements. However, at low force, the situation reversed and the unfolding rate constant decreased with increasing force. Taken together, these data indicate that this small protein has multiple distinct pathways to the native state on the free energy landscape.