Single-molecule FRET dynamics of molecular motors in an ABEL trap

Single-molecule FRET combined with electrokinetic trapping reveals real-time enzyme kinetics of individual F-ATP synthases

“…The presented assay can be adopted for the study of conformational changes in other enzymes as well, as has already been demonstrated for the Rep molecule 34 or RecA binding to ssDNA. 33 Combining single-molecule measurements with long observations times of freely diffusing molecules promises to unravel the dynamics of molecular machines or the folding mechanisms of proteins in the future.…”

“…A purpose-built inverted confocal microscope, augmented with an excitation beam steering unit and controlled by a field-programmable gate array (FPGA) make up the ABEL trap (see ESI† and previous works 19,20,34 for more experimental details of the ABEL trap system). Scanning the excitation laser along a predefined 19-point pattern with a frequency of 5–20 kHz in an ABEL trap multiplies the detection volume of a single laser focus without losing the excellent signal-to-noise characteristics of a confocal system.…”

“…Combining ABEL trapping with smFRET allows to fully exploit the potential of both techniques, particularly for observing conformational transitions in molecular motors and exploring the system's precision near the shot-noise limit. 33 Molecular motors that perform translational or rotational movements benefit from tether-free investigations, for example the Rep molecule, 34 which plays a crucial role in DNA replications, or the F O F 1 -ATP synthase as shown in this work. The advantages of combining smFRET with ABEL trapping are: (1) long observation times of several seconds reveal entire catalysis cycles of individual molecular motors even for slow processes, (2) targeted selection of individual molecules exhibiting FRET increases the data acquisition rate and allows to collect ample single-molecule data for statistical analysis, (3) constant total fluorescence intensity levels during trapping allow to recognize typical single-molecule fingerprints and to exclude anomalies as well as double-labelled molecules, and (4) combining a high temporal resolution with a low pass filter applied to the Fourier-transformed raw data instead of standard time averaging enables us to analyse conformational changes occurring in milliseconds.…”

Correlative approaches in single-molecule biophysics: A review of the progress in methods and applications

“…Steven Quinn and researchers have correlated ratiometric imaging of a reporter dye to determine Ca 2+ concentrations inside model lipid vesicles, with stepwise single-molecule photobleaching of membrane-integrated molecules in aggregated amyloid protein implicated in dementia, to determine the dependence between their molecular stoichiometry and pore formation in the vesicles (35). Maria Dienerowitz and co-workers have utilised an Anti-Brownian ELectrokinetic Trap (ABEL trap) to combine smFRET with molecular confinement to enable very extended observation times of up to several seconds for a key DNA binding protein involved in DNA replication, to explore its multiple molecular conformation states (36). The team of Johannes Holbein have developed phasor-based single-molecule localization microscopy to engineer the point spread function (PSF) shape of single fluorescent reporter dye molecules, integrating hardware modules for deformable mirror control with software for analysing the PSF properties (37).…”

Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution

“…We have built different ABEL traps with APD detectors using a of lasers and associated optical filter sets 13 . Here, the beam diameter of a continuous-wave 532 nm laser (Compass 315M, Coherent) was collimated and adjusted to less than 1 mm by a two lens telescope, and linearly polarized by a polarizing beam splitter cube 14 . Laser power was set to 40 µM using a variable metallic neutral density filter.…”