The Thermodynamic Fingerprints of Ultra-Tight Nanobody-Antigen Binding Probed via Two-Color Single-Molecule Coincidence Detection.

Life on the molecular scale is based on a versatile interplay of biomolecules, a feature that is relevant for the formation of macromolecular complexes. Fluorescence-based two-color coincidence detection is widely used to characterize molecular binding and was recently improved by a brightness-gated version which gives more accurate results. We developed and established protocols which make use of coincidence detection to quantify binding fractions between interaction partners labeled with fluorescence dyes of different colors. Since the applied technique is intrinsically related to single-molecule detection, the concentration of diffusing molecules for confocal detection is typically in the low picomolar regime. This makes the approach a powerful tool for determining bi-molecular binding affinities, in terms of KD values, in this regime. We demonstrated the reliability of our approach by analyzing very strong nanobody-EGFP binding. By measuring the affinity at different temperatures, we were able to determine the thermodynamic parameters of the binding interaction. The results show that the ultra-tight binding is dominated by entropic contributions.

Features of Protein Unfolding Transitions and Their Relation to Domain Topology Probed by Single-Molecule FRET.

A protein fold is defined as a structural arrangement of a secondary structure in a three-dimensional space. It would be interesting to know whether a particular fold can be assigned to certain features of the corresponding folding/unfolding transitions. To understand the underlying principles of the manifold folding transitions in more detail, single-molecule FRET is the method of choice. Taking the two-domain protein phosphoglycerate kinase (PGK) as an example, we investigated denaturant-induced unfolded states of PGK using the above method. For this purpose, different intramolecular distances within the two domains were measured. In addition to the known two-state transition, a transition with a compact folding intermediate was also identified in each of the two domains. Based on the structural homology of the domains (characterized by a Rossmann fold) and the striking similarity in the features of the measured distance changes during unfolding, clear evidence emerged that the underlying domain topology plays an important role in determining the observed structural changes.

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.

Macromolecular Crowding: How Shape and Interactions Affect Diffusion.

A significant fraction of the cell volume is occupied by various proteins, polysaccharides, nucleic acids, etc., which considerably reduces the mobility of macromolecules. Theoretical and experimental work so far have mainly focused on the dependence of the mobility on the occupied volume, while the effect of a macromolecular shape received less attention. Herein, using fluorescence correlation spectroscopy (FCS) and Brownian dynamics (BD) simulations, we report on a dramatic slowdown of tracer diffusion by cylindrically shaped double-stranded (ds) DNAs (16 nm in length). We find, for instance, that the translational diffusion coefficient of a streptavidin tracer is reduced by about 60% for a volume fraction of dsDNA as low as just 5%. For comparison, for a spherical crowder (Ficoll70) the slowdown is only 10% at the same volume fraction and 60% reduction occurs at a volume fraction as high as 35%. BD simulations reveal that this reduction can be attributed to a larger volume excluded to a tracer by dsDNA particles, as compared with spherical Ficoll70 at the same volume fraction, and to the differences in the tracer-crowder attractive interactions. In addition, we find using BD simulations that rotational diffusion of dsDNA is less affected by the crowder shape than its translational motion. Our results show that diffusion in crowded systems is determined not merely by the occupied volume fraction, but that the shape and interactions can determine diffusion, which is relevant to the diverse intracellular environments inside living cells.

Thermophoresis: The Case of Streptavidin and Biotin.

Thermophoretic behavior of a free protein changes upon ligand binding and gives access to information on the binding constants. The Soret effect has also been proven to be a promising tool to gain information on the hydration layer, as the temperature dependence of the thermodiffusion behavior is sensitive to solute-solvent interactions. In this work, we perform systematic thermophoretic measurements of the protein streptavidin (STV) and of the complex STV with biotin (B) using thermal diffusion forced Rayleigh scattering (TDFRS). Our experiments show that the temperature sensitivity of the Soret coefficient is reduced for the complex compared to the free protein. We discuss our data in comparison with recent quasi-elastic neutron scattering (QENS) measurements. As the QENS measurement has been performed in heavy water, we perform additional measurements in water/heavy water mixtures. Finally, we also elucidate the challenges arising from the quantiative thermophoretic study of complex multicomponent systems such as protein solutions.

Transition between protein-like and polymer-like dynamic behavior: Internal friction in unfolded apomyoglobin depends on denaturing conditions.

Equilibrium dynamics of different folding intermediates and denatured states is strongly connected to the exploration of the conformational space on the nanosecond time scale and might have implications in understanding protein folding. For the first time, the same protein system apomyoglobin has been investigated using neutron spin-echo spectroscopy in different states: native-like, partially folded (molten globule) and completely unfolded, following two different unfolding paths: using acid or guanidinium chloride (GdmCl). While the internal dynamics of the native-like state can be understood using normal mode analysis based on high resolution structural information of myoglobin, for the unfolded and even for the molten globule states, models from polymer science are employed. The Zimm model accurately describes the slowly-relaxing, expanded GdmCl-denaturated state, ignoring the individuality of the different aminoacid side chain. The dynamics of the acid unfolded and molten globule state are similar in the framework of the Zimm model with internal friction, where the chains still interact and hinder each other: the first Zimm relaxation time is as large as the internal friction time. Transient formation of secondary structure elements in the acid unfolded and presence of α-helices in the molten globule state lead to internal friction to a similar extent.

Mapping Multiple Distances in a Multidomain Protein for the Identification of Folding Intermediates.

The investigation and understanding of the folding mechanism of multidomain proteins is still a challenge in structural biology. The use of single-molecule Förster resonance energy transfer offers a unique tool to map conformational changes within the protein structure. Here, we present a study following denaturant-induced unfolding transitions of yeast phosphoglycerate kinase by mapping several inter- and intradomain distances of this two-domain protein, exhibiting a quite heterogeneous behavior. On the one hand, the development of the interdomain distance during the unfolding transition suggests a classical two-state unfolding behavior. On the other hand, the behavior of some intradomain distances indicates the formation of a compact and transient molten globule intermediate state. Furthermore, different intradomain distances measured within the same domain show pronounced differences in their unfolding behavior, underlining the fact that the choice of dye attachment positions within the polypeptide chain has a substantial impact on which unfolding properties are observed by single-molecule Förster resonance energy transfer measurements. Our results suggest that, to fully characterize the complex folding and unfolding mechanism of multidomain proteins, it is necessary to monitor multiple intra- and interdomain distances because a single reporter can lead to a misleading, partial, or oversimplified interpretation.

Strong Adverse Contribution of Conformational Dynamics to Streptavidin-Biotin Binding.

Molecular dynamics plays an important role for the biological function of proteins. For protein ligand interactions, changes of conformational entropy of protein and hydration layer are relevant for the binding process. Quasielastic neutron scattering (QENS) was used to investigate differences in protein dynamics and conformational entropy of ligand-bound and ligand-free streptavidin. Protein dynamics were probed both on the fast picosecond time scale using neutron time-of-flight spectroscopy and on the slower nanosecond time scale using high-resolution neutron backscattering spectroscopy. We found the internal equilibrium motions of streptavidin and the corresponding mean square displacements (MSDs) to be greatly reduced upon biotin binding. On the basis of the observed MSDs, we calculated the difference of conformational entropy ΔSconf of the protein component between ligand-bound and ligand-free streptavidin. The rather large negative ΔSconf value (-2 kJ mol-1 K-1 on the nanosecond time scale) obtained for the streptavidin tetramer seems to be counterintuitive, given the exceptionally high affinity of streptavidin-biotin binding. Literature data on the total entropy change ΔS observed upon biotin binding to streptavidin, which includes contributions from both the protein and the hydration water, suggest partial compensation of the unfavorable ΔSconf by a large positive entropy gain of the surrounding hydration layer and water molecules that are displaced during ligand binding.

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 Techniques and Cell-Free Protein Synthesis: A Perfect Marriage.

Single-molecule techniques are currently an essential tool to study conformational changes as well as the synthesis and folding of proteins. However, the preparation of suitable protein samples is often time-consuming and demanding. The rapid development of cell-free protein synthesis over the last few years opened new perspectives for fast and easy sample preparation, but this was not fully exploited until now. Here, we take a look at the advancements in sample preparation as well as in the development of technical approaches and analytical tools, which unavoidably lead to the combination of single-molecule techniques and cell-free protein synthesis. It is an ideal combination that can unlock the full potential of studying complex biological processes in the near future.

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.

Cotranslational Incorporation into Proteins of a Fluorophore Suitable for smFRET Studies.

Single-molecule FRET (smFRET) is a powerful tool to investigate conformational changes of biological molecules. In general, smFRET studies require protein samples that are site-specifically double-labeled with a pair of donor and acceptor fluorophores. The common approaches to produce such samples cannot be applied when studying the synthesis and folding of the polypeptide chain on the ribosome. The best strategy is to incorporate two fluorescent amino acids cotranslationally using cell-free protein synthesis systems. Here, we demonstrate the cotranslational site-specific incorporation into a model protein of Atto633, a dye with excellent photophysical properties, suitable for single molecule spectroscopy, together with a second dye using a combination of the sense cysteine and the nonsense amber codon. In this work we show that cotranslational incorporation of good fluorophores into proteins is a viable strategy to produce suitable samples for smFRET studies.

Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies.

Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET studies. The protocol describes two cell-free approaches to achieve a selective label scheme that allows the highest possible accuracy in inter-dye distance determination.

Selective Double-Labeling of Cell-Free Synthesized Proteins for More Accurate smFRET Studies.

Förster resonance energy transfer (FRET) studies performed at the single molecule level have unique abilities to probe molecular structure, dynamics, and function of biological molecules. This technique requires specimens, like proteins, equipped with two different fluorescent probes attached at specific positions within the molecule of interest. Here, we present an approach of cell-free protein synthesis (CFPS) that provides proteins with two different functional groups for post-translational labeling at the specific amino acid positions. Besides the sulfhydryl group of a cysteine, we make use of an azido group of a p-azido-l-phenylalanine to achieve chemical orthogonality. Herein, we achieve not only a site-specific but, most importantly, also a site-selective, label scheme that permits the highest accuracy of measured data. This is demonstrated in a case study, where we synthesize human calmodulin (CaM) by using a CFPS kit and prove the structural integrity and the full functionality of this protein.

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.

Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions.

The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye-dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil-globule transition.

In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.