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.

High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization.

Many viruses form highly pleomorphic particles. In influenza, virion structure is of interest not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline, a method well-suited to the study of large numbers of pleomorphic structures, to image many thousands of individual influenza virions; gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super-resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. We also showed that viral RNP complexes are located preferentially within Archetti bodies when these were observed at filament ends, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses.

The fork protection complex recruits FACT to reorganize nucleosomes during replication.

Chromosome replication depends on efficient removal of nucleosomes by accessory factors to ensure rapid access to genomic information. Here, we show this process requires recruitment of the nucleosome reorganization activity of the histone chaperone FACT. Using single-molecule FRET, we demonstrate that reorganization of nucleosomal DNA by FACT requires coordinated engagement by the middle and C-terminal domains of Spt16 and Pob3 but does not require the N-terminus of Spt16. Using structure-guided pulldowns, we demonstrate instead that the N-terminal region is critical for recruitment by the fork protection complex subunit Tof1. Using in vitro chromatin replication assays, we confirm the importance of these interactions for robust replication. Our findings support a mechanism in which nucleosomes are removed through the coordinated engagement of multiple FACT domains positioned at the replication fork by the fork protection complex.

Structural dynamics in the evolution of a bilobed protein scaffold.

Novel biophysical tools allow the structural dynamics of proteins and the regulation of such dynamics by binding partners to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remain poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the periplasmic-binding protein-like II domain family, have undergone divergent evolution, leading to adaptation of their structural dynamics. We performed a structural analysis on ∼600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Förster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enable distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extracytoplasmic transport-related proteins. Structural embellishments of the core created interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately altered substrate specificity. Our findings reveal an as-yet-unrecognized mechanism for the emergence of functional promiscuity during long periods of evolution and are applicable to a large number of domain architectures.

Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold.

SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5 µM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5 µM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.

Conformational dynamics of the ABC transporter McjD seen by single-molecule FRET.

ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward-open conformation) and hydrolysis (inward-open) for unidirectional transport driven by ATP Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single-molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native-like lipid environment and directly monitor conformational dynamics in both the transmembrane- (TMD) and nucleotide-binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.

Linker Molecules Convert Commercial Fluorophores into Tailored Functional Probes during Biolabelling.

Many life-science techniques and assays rely on selective labeling of biological target structures with commercial fluorophores that have specific yet invariant properties. Consequently, a fluorophore (or dye) is only useful for a limited range of applications, e.g., as a label for cellular compartments, super-resolution imaging, DNA sequencing or for a specific biomedical assay. Modifications of fluorophores with the goal to alter their bioconjugation chemistry, photophysical or functional properties typically require complex synthesis schemes. We here introduce a general strategy that allows to customize these properties during biolabelling with the goal to introduce the fluorophore in the last step of biolabelling. For this, we present the design and synthesis of 'linker' compounds, that bridge biotarget, fluorophore and a functional moiety via well-established labeling protocols. Linker molecules were synthesized via the Ugi four-component reaction (Ugi-4CR) which facilitates a modular design of linkers with diverse functional properties and bioconjugation- and fluorophore attachment moieties. To demonstrate the possibilities of different linkers experimentally, we characterized the ability of commercial fluorophores from the classes of cyanines, rhodamines, carbopyronines and silicon-rhodamines to become functional labels on different biological targets in vitro and in vivo via thiol-maleimide chemistry. With our strategy, we showed that the same commercial dye can become a photostable self-healing dye or a sensor for bivalent ions subject to the linker used. Finally, we quantified the photophysical performance of different self-healing linker-fluorophore conjugates and demonstrated their applications in super-resolution imaging and single-molecule spectroscopy.

Characterization of Fluorescent Proteins with Intramolecular Photostabilization*.

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet-state quenchers or redox-active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the ß-barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series.

The front cover artwork was provided by Gabriel Moya, a PhD student in the Cordes lab (LMU Biocenter Munich, Germany). The cover image shows an artistic impression of the chemical structures of two fluorophores investigated in the paper. Read the full text of the Article at 10.1002/cphc.202000935.

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series*.

The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.

Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications*.

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.

Single-Molecule Observation of Ligand Binding and Conformational Changes in FeuA.

The specific binding of ligands by proteins and the coupling of this process to conformational changes is fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ATP-binding cassette importer FeuBC, which is involved in bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer, whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10,000-fold and temporally stabilize the formed conformation 250-fold.

On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores.

While buffer cocktails remain the most commonly used method for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with 'self-healing' or even functional properties such as photoswitching. In this contribution, we evaluated combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other. We find that although the rate of triplet-state quenching is additive, the photostability is limited by the faster pathway. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. Furthermore we show that intramolecular photostabilizers can protect fluorophores from reversible off-switching events caused by solution-additives, which was previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer-dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics in different biochemical environments.

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA.

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments.

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor-acceptor pairs during solution-based smFRET. We use this "caged FRET" methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.

Watching conformational dynamics of ABC transporters with single-molecule tools.

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

Intramolecular photostabilization via triplet-state quenching: design principles to make organic fluorophores "self-healing".

Covalent linkage of fluorophores and photostabilizers was recently revived as a strategy to make organic fluorophores "self-healing"via triplet-state quenching. Although Lüttke and co-workers pioneered this strategy already in the 1980s, the general design principles still remain elusive. In this contribution, we combine experiments and theory to understand what determines the photostabilization efficiency in dye-photostabilizer conjugates. Our results from single-molecule microscopy and molecular dynamics simulations of different Cy5-derivatives suggest that the distance and relative geometry between the fluorophore and photostabilizer are more important than the chemical nature of the photostabilizer, e.g. its redox potential, which is known to influence electron-transfer rates. We hypothesize that the efficiency of photostabilization scales directly with the contact rate of the fluorophore and photostabilizer. This study represents an important step in the understanding of the molecular mechanism of intramolecular photostabilization and can pave the way for further development of stable emitters for various applications.