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

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories 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 with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.

Coherent Doppler wind lidar with real-time wind processing and low signal-to-noise ratio reconstruction based on a convolutional neural network.

Multi-classification using a convolutional neural network (CNN) is proposed as a denoising method for coherent Doppler wind lidar (CDWL) data. The method is intended to enhance the usable range of a CDWL beyond the atmospheric boundary layer (ABL). The method is implemented and tested in an all-fiber pulsed CWDL system operating at 1550 nm wavelength with 20 kHz repetition rate, 300 ns pulse length and 180 µJ of laser energy. Real-time pre-processing using a field programmable gate array (FPGA) is implemented producing averaged lidar spectrograms. Real-world measurement data is labeled using conventional frequency estimators and mixed with simulated spectrograms for training of the CNN. First results of this methods show that the CNN can outperform conventional frequency estimations substantially in terms of maximum range and delivers reasonable output in very low signal-to-noise (SNR) situations while still delivering accurate results in the high-SNR regime. Comparing the CNN output with radiosonde data shows the feasibility of the proposed method.

Inside a Shell-Organometallic Catalysis Inside Encapsulin Nanoreactors.

Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self-assembling protein-based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single-molecule Förster resonance energy transfer and 3D-MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single-molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non-native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space.

Temperature dependence of the conversion efficiency of photochromic perylene bisimide dithienylcyclopentene triads embedded in a polymer.

Photochromic molecules that are covalently linked to a strong fluorophore combine the requirements of external control and strong fluorescence, which will become increasingly important for super-resolution microscopy techniques based on single molecules. However, given the bulky structure of such constructs, steric hindrance might affect their photoconversion efficiencies upon immobilising them for imaging purposes. In this study the efficiencies of the photochromic conversion processes of molecular triads that are embedded in a polymer have been studied as a function of temperature. The triads consist of two perylene bisimide dye molecules that are connected via a dithienylcyclopentene photochromic bridge that undergoes a cyclization/cycloreversion reaction upon appropriate illumination. It is found that photochromic switching remains active, even at 5 K, yet with reduced but finite efficiency for the cycloreversion reaction. This might even be an advantage for the achievement of high labelling densities in super-resolution microscopy.

Hierarchical dynamics in allostery following ATP hydrolysis monitored by single molecule FRET measurements and MD simulations.

We report on a study that combines advanced fluorescence methods with molecular dynamics (MD) simulations to cover timescales from nanoseconds to milliseconds for a large protein. This allows us to delineate how ATP hydrolysis in a protein causes allosteric changes at a distant protein binding site, using the chaperone Hsp90 as test system. The allosteric process occurs via hierarchical dynamics involving timescales from nano- to milliseconds and length scales from Ångstroms to several nanometers. We find that hydrolysis of one ATP is coupled to a conformational change of Arg380, which in turn passes structural information via the large M-domain α-helix to the whole protein. The resulting structural asymmetry in Hsp90 leads to the collapse of a central folding substrate binding site, causing the formation of a novel collapsed state (closed state B) that we characterise structurally. We presume that similar hierarchical mechanisms are fundamental for information transfer induced by ATP hydrolysis through many other proteins.

Kinetics of Transient Protein Complexes Determined via Diffusion-Independent Microfluidic Mixing and Fluorescence Stoichiometry.

Low-affinity protein complexes and their transient states are difficult to measure in single-molecule experiments because of their low population at low concentrations. A prominent solution to this problem is the use of microfluidic mixing devices, which rely on diffusion-based mixing. This is not ideal for multiprotein complexes, as the single-molecule fluorescence signal is dominated by the already dissociated species. Here, we designed a microfluidic device with mixing structures for fast and homogeneous mixing of components with varying diffusion coefficients and for fluorescence measurements at a defined single-molecule concentration. This enables direct measurement of dissociation rates at a broad range of timescales from a few milliseconds to several minutes. This further allows us to measure structural properties and stoichiometries of protein complexes with large equilibrium dissociation constants ( KD's) of 5 µM and above. We used the platform to measure structural properties and dissociation rates of heat shock protein 90 (Hsp90) dimers and found at least two dissociation rates which depend on the nucleotide state. Finally, we demonstrate the capability for measuring also equilibrium dissociation constants, resulting in the determination of both the kinetics and thermodynamics of the system under investigation.

A photoswitchable poly(3-hexylthiophene).

A well-defined main-chain conjugated photoswitchable poly(3-hexylthiophene) with homogeneous hydrogen end groups was synthesized. Opening and closing the photoswitch enables reversible modulation of P3HT emission, which is quenched by 70% when the switch is closed. The optical properties during switching cycles were quantified by irradiation/spectroscopy sequences.