Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution.

For single-molecule studies in solution, very small concentrations of dye-labelled molecules are employed in order to achieve single-molecule sensitivity. In typical studies with confocal microscopes, often concentrations in the pico-molar regime are required. For various applications that make use of single-molecule Förster resonance energy transfer (smFRET) or two-color coincidence detection (TCCD), the molecule concentration must be set explicitly to targeted values and furthermore needs to be stable over a period of several hours. As a consequence, specific demands must be imposed on the surface passivation of the cover slides during the measurements. The aim of having only one molecule in the detection volume at the time is not only affected by the absolute molecule concentration, but also by the rate of diffusion. Therefore, we discuss approaches to control and to measure absolute molecule concentrations. Furthermore, we introduce an approach to calculate the probability of chance coincidence events and demonstrate that measurements with challenging smFRET samples require a strict limit of maximal sample concentrations in order to produce meaningful results.

Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations.

Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein.

Distinct pre-initiation steps in human mitochondrial translation.

Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.

Brightness-gated two-color coincidence detection unravels two distinct mechanisms in bacterial protein translation initiation.

Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial. Fluorescence based two-color coincidence detection (TCCD) is commonly used to characterize molecular binding, but suffers from an underestimation of coincident events. Here, we introduce a brightness-gated TCCD which overcomes this limitation and benchmark our approach with two custom-made calibration samples. Applied to a cell-free protein synthesis assay, brightness-gated TCCD unraveled a previously disregarded mode of translation initiation in bacteria.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules.

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa. Polyethylene glycol and Ficoll polymers of different molecular masses were used in buffer solutions to mimic a crowded environment. The reduction in translational mobility of the biological tracer molecules was analyzed as a function of crowder volume fractions and was generally more pronounced in PEG as compared to Ficoll solutions. For several crowding conditions, we observed a molecular sieving effect, in which the diffusion coefficient of larger tracer molecules is reduced to a larger extent than predicted by the Stokes-Einstein relation. By employing a FRET-based biosensor, we also showed that a multiprotein complex is significantly compacted in the presence of macromolecular crowders. Importantly, with respect to sensor in vivo applications, ligand concentration determining sensors would need a crowding specific calibration in order to deliver correct cytosolic ligand concentration.

Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species.

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

Genetically Encoded Förster Resonance Energy Transfer-Based Biosensors Studied on the Single-Molecule Level.

Genetically encoded Förster resonance energy transfer (FRET)-based biosensors for the quantification of ligand molecules change the magnitude of FRET between two fluorescent proteins upon binding a target metabolite. When highly sensitive sensors are being designed, extensive sensor optimization is essential. However, it is often difficult to verify the ideas of modifications made to a sensor during the sensor optimization process because of the limited information content of ensemble FRET measurements. In contrast, single-molecule detection provides detailed information and higher accuracy. Here, we investigated a set of glucose and crowding sensors on the single-molecule level. We report the first comprehensive single-molecule study of FRET-based biosensors with reasonable counting statistics and identify characteristics in the single-molecule FRET histograms that constitute fingerprints of sensor performance. Hence, our single-molecule approach extends the toolbox of methods aiming to understand and optimize the design of FRET-based biosensors.

A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems.

Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separately quantify the two main components of productivity, namely the fraction of active ribosomes and the number of synthesizing cycles. Here, we provide a direct and highly reliable measure of ribosomal activity in any given CFPS system, introducing an enhanced-arrest peptide variant. We observe an almost complete stalling of ribosomes that produce GFPem (~95%), as determined by common centrifugation techniques and fluorescence correlation spectroscopy (FCS). Moreover, we thoroughly study the effect of different ribosomal modifications independently on activity and number of synthesizing cycles. Finally, employing two-colour coincidence detection and two-colour colocalisation microscopy, we demonstrate real-time access to key productivity parameters with minimal sample consumption on a single ribosome level.

Inter-dye distance distributions studied by a combination of single-molecule FRET-filtered lifetime measurements and a weighted accessible volume (wAV) algorithm.

Förster resonance energy transfer (FRET) is an important tool for studying the structural and dynamical properties of biomolecules. The fact that both the internal dynamics of the biomolecule and the movements of the biomolecule-attached dyes can occur on similar timescales of nanoseconds is an inherent problem in FRET studies. By performing single-molecule FRET-filtered lifetime measurements, we are able to characterize the amplitude of the motions of fluorescent probes attached to double-stranded DNA standards by means of flexible linkers. With respect to previously proposed experimental approaches, we improved the precision and the accuracy of the inter-dye distance distribution parameters by filtering out the donor-only population with pulsed interleaved excitation. A coarse-grained model is employed to reproduce the experimentally determined inter-dye distance distributions. This approach can easily be extended to intrinsically flexible proteins allowing, under certain conditions, to decouple the macromolecule amplitude of motions from the contribution of the dye linkers.