An automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics.

Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein-DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.

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.

Recruiting Unicellular Algae for the Mass Production of Nanostructured Perovskites.

Functional capacities of lead halide perovskites are strongly dependent on their morphology, crystallographic texture, and internal ultrastructure on the nano- and the meso-scale. In the last decade, significant efforts are directed towards the development of novel synthesis routes that would overcome the morphological constraints provided by the physical and crystallographic properties of these materials. In contrast, various living organisms, such as unicellular algae, have the ability to mold biogenic crystals into a vast variety of intricate nano-architectured shapes while keeping their single crystalline nature. Here, using the cell wall of the dinoflagellate L. granifera as a model, sustainably harvested biogenic calcite is successfully transformed into nano-structured perovskites. Three variants of lead halide perovskites CH3 NH3 PbX3 are generated with X = Cl- , Br- and I- ; exhibiting emission peak-wavelength ranging from blue, to green, to near-infrared, respectively. The approach can be used for the mass production of nano-architectured perovskites with desired morphological, textural and, consequently, physical properties exploiting the numerous templates provided by calcite forming unicellular organisms.

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins.

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such 'normalization' to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

Single-molecule approaches reveal outer membrane protein biogenesis dynamics.

Outer membrane proteins (OMPs) maintain the viability of Gram-negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi-protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single-molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis.

Evidence for endogenous exchange of cytoplasmic material between a subset of cone and rod photoreceptors within the adult mammalian retina via direct cell-cell connections.

Photoreceptor cell transplantation into the mouse retina has been shown to result in the transfer of cytoplasmic material between donor and host photoreceptors. Recently it has been found that this inter-photoreceptor material transfer process is likely to be mediated by nanotube-like structures connecting donor and host photoreceptors. By leveraging cone-specific reporter mice and super-resolution microscopy we provide evidence for the transfer of cytoplasmic material also from endogenous cones to endogenous rod photoreceptors and the existence of nanotube-like cell-cell connections possibly mediating this process in the adult mouse retina, together with preliminary data indicating that horizontal material transfer may also occur in the human retina.

Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates.

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs.

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α-helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute-timescale heat treatment of Trx-tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single-step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

Fast, Simultaneous Tagging and Mutagenesis of Genes on Bacterial Chromosomes.

Fluorescence microscopy has become a powerful tool in molecular cell biology. Visualizing specific proteins in bacterial cells requires labeling with fluorescent or fluorogenic tags, preferentially at the native chromosomal locus to preserve expression dynamics associated with the genomic environment. Exploring protein function calls for targeted mutagenesis and observation of differential phenotypes. In the model bacterium Escherichia coli, protocols for tagging genes and performing targeted mutagenesis currently involve multiple steps. Here, we present an approach capable of simultaneous tagging and mutagenesis of essential and nonessential genes in a single step. We require only the insertion of a stretch of the target gene into an auxiliary plasmid together with the tag. Recombineering-based exchange with the native locus is then carried out, where the desired mutation is introduced during amplification with homology-bearing primers. Using this approach, multiple tagged mutants per gene can be derived quickly.

Towards next generation therapies for cystic fibrosis: Folding, function and pharmacology of CFTR.

The treatment of cystic fibrosis (CF) has been transformed by orally-bioavailable small molecule modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), which restore function to CF mutants. However, CFTR modulators are not available to all people with CF and better modulators are required to prevent disease progression. Here, we review selectively recent advances in CFTR folding, function and pharmacology. We highlight ensemble and single-molecule studies of CFTR folding, which provide new insight into CFTR assembly, its perturbation by CF mutations and rescue by CFTR modulators. We discuss species-dependent differences in the action of the F508del-CFTR mutation on CFTR expression, stability and function, which might influence pharmacological studies of CFTR modulators in CF animal models. Finally, we illuminate the identification of combinations of two CFTR potentiators (termed co-potentiators), which restore therapeutically-relevant levels of CFTR activity to rare CF mutations. Thus, mechanistic studies of CFTR folding, function and pharmacology inform the development of highly effective CFTR modulators.

SDS-induced multi-stage unfolding of a small globular protein through different denatured states revealed by single-molecule fluorescence.

Ionic surfactants such as sodium dodecyl sulfate (SDS) unfold proteins in a much more diverse yet effective way than chemical denaturants such as guanidium chloride (GdmCl). But how these unfolding processes compare on a molecular level is poorly understood. Here, we address this question by scrutinising the unfolding pathway of the globular protein S6 in SDS and GdmCl with single-molecule Förster resonance energy transfer (smFRET) spectroscopy. We show that the unfolding mechanism in SDS is strikingly different and convoluted in comparison to denaturation in GdmCl. In contrast to the reversible two-state unfolding behaviour in GdmCl characterised by kinetics on the timescale of seconds, SDS demonstrated not one, but four distinct regimes of interactions with S6, dependent on the surfactant concentration. At ≤1 mM SDS, S6 and surfactant molecules form quasi-micelles on a minute timescale; at millimolar [SDS], the protein denatures through an unfolded/denatured ensemble of highly heterogeneous states on a multi-second timescale; at tens of millimolar of SDS, the protein unfolds into a micelle-packed conformation on the second timescale; and >50 mM SDS, the protein unfolds with millisecond timescale dynamics. We propose a detailed model for multi-stage unfolding of S6 in SDS, which involves at least three different types of denatured states with different level of compactness and dynamics and a continually changing landscape of interactions between protein and surfactant. Our results highlight the great potential of single-molecule fluorescence as a direct probe of nanoscale protein structure and dynamics in chemically complex surfactant environments.

CFTR transmembrane segments are impaired in their conformational adaptability by a pathogenic loop mutation and dynamically stabilized by Lumacaftor.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel protein that is defective in individuals with cystic fibrosis (CF). To advance the rational design of CF therapies, it is important to elucidate how mutational defects in CFTR lead to its impairment and how pharmacological compounds interact with and alter CFTR. Here, using a helical-hairpin construct derived from CFTR's transmembrane (TM) helices 3 and 4 (TM3/4) and their intervening loop, we investigated the structural effects of a patient-derived CF-phenotypic mutation, E217G, located in the loop region of CFTR's membrane-spanning domain. Employing a single-molecule FRET assay to probe the folding status of reconstituted hairpins in lipid bilayers, we found that the E217G hairpin exhibits an altered adaptive packing behavior stemming from an additional GXXXG helix-helix interaction motif created in the mutant hairpin. This observation suggested that the misfolding and functional defects caused by the E217G mutation arise from an impaired conformational adaptability of TM helical segments in CFTR. The addition of the small-molecule corrector Lumacaftor exerts a helix stabilization effect not only on the E217G mutant hairpin, but also on WT TM3/4 and other mutations in the hairpin. This finding suggests a general mode of action for Lumacaftor through which this corrector efficiently improves maturation of various CFTR mutants.

Enhancing the stability of DNA origami nanostructures: staple strand redesign versus enzymatic ligation.

DNA origami structures have developed into versatile tools in molecular sciences and nanotechnology. Currently, however, many potential applications are hindered by their poor stability, especially under denaturing conditions. Here we present and evaluate two simple approaches to enhance DNA origami stability. In the first approach, we elevated the melting temperature of nine critical staple strands by merging the oligonucleotides with adjacent sequences. In the second approach, we increased the global stability by enzymatically ligating all accessible staple strand ends directly. By monitoring the gradual urea-induced denaturation of a prototype triangular DNA origami modified by these approaches using atomic force microscopy, we show that rational redesign of a few, critical staple strands leads to a considerable increase in overall stability at high denaturant concentration and elevated temperatures. In addition, enzymatic ligation yields DNA nanostructures with superior stability at up to 37 °C and in the presence of 6 M urea without impairing their shape. This bio-orthogonal approach is readily adaptable to other DNA origami structures without the need for synthetic nucleotide modifications when structural integrity under harsh conditions is required.

Structural dynamics of membrane-protein folding from single-molecule FRET.

Membrane proteins fulfil a plethora of vital functions, are major drug targets, and are implicated in many diseases. Their importance, however, is in no way paralleled by our current understanding of the dynamic processes by which these proteins fold into and function within cellular membranes. This is mainly due to fundamental challenges in resolving the structural dynamics of proteins embedded within lipid-bilayer membranes or membrane-mimetic environments. Single-molecule spectroscopy bears great potential for dissecting this complexity. Particularly, single-molecule Förster resonance energy transfer (smFRET), owing to its sensitivity and versatility, has emerged as a new tool for accessing the spatial, temporal, and energetic features of membrane-protein folding reactions, providing unique insights into protein subpopulations and their associated dynamics on timescales ranging from nanoseconds to hours. Here, we review recent advances in the application of smFRET to the structural dynamics of membrane-protein folding and discuss the benefits that this new toolset affords to provide a molecular-level description of the dynamics governing this physiologically and therapeutically eminent class of proteins.

Real-time monitoring of protein-induced DNA conformational changes using single-molecule FRET.

Apart from being storage devices for genetic information, nucleic acids can provide regulatory structures through evolutionarily optimized sequences. The interaction of proteins binding specifically to such sequences and resulting secondary structures, or the exposure of single-stranded DNA add a versatile regulatory framework for cells. Biochemical and structural biology experiments have revealed important underlying concepts of protein-DNA interactions but are often limited by ensemble averaging or static information. To decipher the dynamics of conformations adopted by protein-DNA complexes, single-molecule approaches have become a powerful resource over the past two decades. In particular single-molecule FRET (smFRET), which allows a read-out of DNA or protein conformations, became widely used. Here, we illustrate how to implement the technique and exemplarily describe how smFRET yields insights into conformational changes of DNA secondary structures induced by the single-stranded DNA binding protein SSB. We further explain how we use smFRET to study mechanisms of the replication initiator DnaA and the competition of DnaA and SSB for single-stranded DNA. We anticipate that smFRET will further develop into a particularly useful technique to study dynamic competitions of proteins for the same DNA substrate.

Hexameric helicase G40P unwinds DNA in single base pair steps.

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

Structural heterogeneity of attC integron recombination sites revealed by optical tweezers.

A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins-so-called attC sites-with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom (${{att}}{{{C}}_{{\rm{bs}}}}$) and top (${{att}}{{{C}}_{{\rm{ts}}}}$) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures-a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred ${{att}}{{{C}}_{{\rm{bs}}}}$ predominantly formed the straight hairpin, while the ${{att}}{{{C}}_{{\rm{ts}}}}$ preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the ${{att}}{{{C}}_{{\rm{ts}}}}$in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins.

A minimal helical-hairpin motif provides molecular-level insights into misfolding and pharmacological rescue of CFTR.

Our meagre understanding of CFTR misfolding and its reversal by small-molecule correctors hampers the development of mechanism-based therapies of cystic fibrosis. Here we exploit a helical-hairpin construct-the simplest proxy of membrane-protein tertiary contacts-containing CFTR's transmembrane helices 3 and 4 and its corresponding disease phenotypic mutant V232D to gain molecular-level insights into CFTR misfolding and drug rescue by the corrector Lumacaftor. Using a single-molecule FRET approach to study hairpin conformations in lipid bilayers, we find that the wild-type hairpin is well folded, whereas the V232D mutant assumes an open conformation in bilayer thicknesses mimicking the endoplasmic reticulum. Addition of Lumacaftor reverses the aberrant opening of the mutant hairpin to restore a compact state as in the wild type. The observed membrane escape of the V232D hairpin and its reversal by Lumacaftor complement cell-based analyses of the full-length protein, thereby providing in vivo and in vitro correlates of CFTR misfolding and drug-action mechanisms.

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.