ABEL-FRET: tether-free single-molecule FRET with hydrodynamic profiling

Abstract

Single-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental protocols often resort to molecule immobilization for long observation times and rarely approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with near shot-noise limited, Angstrom-level resolution in FRET efficiency. Furthermore, ABEL-FRET naturally integrates hydrodynamic profiling, which harnesses single-molecule diffusion coefficient to enhance FRET sensing of biological processes.

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

“…Accordingly, two significantly different FRET efficiencies (E FRET ) are expected to be distinguishable in time traces of single EF o F 1 during ATP- driven ε-subunit rotation, i.e., E FRET =0.35 for L (or “ medium FRET ”) and E FRET =0.90 for H 1 or E FRET =0.92 for H 2 , respectively. Both H 1 and H 2 are “ high FRET ” and are not distinguishable with our experimental setup (see also 47 ). We simulated FRET traces of single EF o F 1 in an ABEL trap and confirmed that H 1 and H 2 cannot be discriminated, both for short and for long dwell times (for instance see Figure 1 E, simulated with more distinct FRET efficiencies E FRET =0.80 for H 1 and E FRET =0.90 for H 2 ).…”

“…Corrections have to compensate for the spectral transmission of the microscope objective, spectral properties of the optical filters, spectral sensitivity of the APD detectors, and fluorescence quantum yields of the fluorophores 74 . Photon counts of individual FRET-labeled EF o F 1 are maximized by the ABEL trap 47 . High photon counts will allow to precisely identify distinct conformations of active EF o F 1 during turnover.…”

“…This trap is designed to push a fluorescent nanoparticle to the center of the laser focus pattern in real time using matching electric field gradients. After a diffusing single object is trapped, it is hold in place without surface interference 47 . The ABEL trap allows to compare and optimize fluorophores for smFRET 48 .…”

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.…”

“…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.…”