Biology subjects

Chamachi, N.

Publications and source records attributed to Chamachi, N..

3 recordsLinked to original sources

bioRxiv2023

Farewell to single-well: An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics

Single-molecule FRET (smFRET) has become a widely used tool for probing the structure, dynamics, and functional mechanisms of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Investigations by smFRET often require sampling of a large parameter space, for example, by varying one or more constituent molecular components in ten or more steps to reliably extract distances, kinetic rates, and other quantitative parameters. Confocal smFRET measurements, for example, which are amongst the widely used smFRET assays, are typically performed in a single-well format and measurements are conducted in a manual manner, making sampling of many experimental parameters laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality into a confocal microscope to allow for continuous and automated smFRET measurements. We show that the multiwell plate assay is on par with conventional single-well smFRET measurements in terms of accuracy and precision yet enables probing tens to hundreds of conditions in a fully automized manner. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, and competitive and cooperative protein-DNA interactions, revealing new insights that would be hard if not impossible to achieve with conventional single-well format measurements. The higher sampling density afforded by the multiwell plate format increases the accuracy of data analysis by at least 10-fold. We further showcase that the assay provides access to smFRET-based screening of drug-protein interactions. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software. Taken together, the automated multiwell plate assay developed here opens up new possibilities to acquire high-content smFRET datasets for in-depth single-molecule analysis of biomolecular conformations, interactions, and dynamics.

biophysics↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2021

Chaperones Skp and SurA dynamically expand unfolded outer membrane protein X and synergistically disassemble oligomeric aggregates

Periplasmic chaperones Skp and SurA are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA association with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX, and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multi-faceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

biophysics↗