Single-molecule Fluorescence Energy Transfer Research Articles

Homo- and hetero-dimeric subunit interactions set affinity and efficacy in metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are dimeric class C G-protein–coupled receptors that operate in glia and neurons. Glutamate affinity and efficacy vary greatly between the eight mGluRs. The molecular basis of this diversity is not understood. We used single-molecule fluorescence energy transfer to monitor the structural rearrangements of activation in the mGluR ligand binding domain (LBD). In saturating glutamate, group II homodimers fully occupy the activated LBD conformation (full efficacy) but homodimers of group III mGluRs do not. Strikingly, the reduced efficacy of Group III homodimers does not arise from differences in the glutamate binding pocket but, instead, from interactions within the extracellular dimerization interface that impede active state occupancy. By contrast, the functionally boosted mGluR II/III heterodimers lack these interface ‘brakes’ to activation and heterodimer asymmetry in the flexibility of a disulfide loop connecting LBDs greatly favors occupancy of the activated conformation. Our results suggest that dimerization interface interactions generate substantial functional diversity by differentially stabilizing the activated conformation. This diversity may optimize mGluR responsiveness for the distinct spatio-temporal profiles of synaptic versus extrasynaptic glutamate.

Enabling multiperspective single-molecule FRET.

Single-molecule fluorescence studies enable real-time observation of interactions between molecular machines using minuscule amounts of (potentially) precious samples. Experimental repetition is required for optimizations, to test different samples or conditions, and ultimately to perform technical and biological repeats for publication. Here we developed a multiplexed approach that allows us to significantly increase experimental sensitivity, reproducibility, and throughput of single-molecule fluorescence and fluorescence energy transfer imaging. By coupling this approach with nucleic acid barcoding, microfluidic handling systems, and advanced data processing, we aim to automate and simplify multiperspective single-molecule studies to accelerate the emerging field of dynamic structural biology.

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

Single-Molecule FRET Detection of Sub-Nanometer Distance Changes in the Range below a 3-Nanometer Scale.

Single-molecule fluorescence energy transfer (FRET) detection has become a key technique to monitor intra- and intermolecular distance changes in biological processes. As the sensitive detection range of conventional FRET pairs is limited to 3–8 nm, complement probes are necessary for extending this typical working range. Here, we realized a single-molecule FRET assay for a short distance range of below 3 nm by using a Cy2–Cy7 pair having extremely small spectral overlap. Using two DNA duplexes with a small difference in the labeling position, we demonstrated that our assay can observe subtle changes at a short distance range. High sensitivity in the range of 1–3 nm and compatibility with the conventional FRET assay make this approach useful for understanding dynamics at a short distance.

Ultrafast DNA Sensors with DNA Framework-Bridged Hybridization Reactions.

Intracellular DNA-based hybridization reactions generally occur under tension rather than in free states, which are spatiotemporally controlled in physiological conditions. However, how nanomechanical forces affect DNA hybridization efficiencies in in-vitro DNA assays, for example, biosensors or biochips, remains largely elusive. Here, we design DNA framework-based nanomechanical handles that can control the stretching states of DNA molecules. Using a pair of tetrahedral DNA framework (TDF) nanostructured handles, we develop bridge DNA sensors that can capture target DNA with ultrafast speed and high efficiency. We find that the rigid TDF handles bind two ends of a single-stranded DNA (ssDNA) and hold it in a stretched state, with an apparent stretching length comparable to its counterpart of double-stranded DNA (dsDNA) via atomic force microscopy measurement. The DNA stretching effect of ssDNA is then monitored using single-molecule fluorescence energy transfer (FRET), resulting in decreased FRET efficiency in the stretched ssDNA. By controlling the stretching state of ssDNA, we obtained significantly improved hybridization kinetics (within 1 min) and hybridization efficiency (∼98%) under the target concentration of 500 nM. The bridge DNA sensors demonstrated high sensitivity (1 fM), high specificity (single mismatch mutation discrimination), and high selectivity (suitable for the detection in serum and blood) under the target concentration of 10 nM. Controlling the stretching state of ssDNA shows great potential in biosensors, bioimaging, and biochips applications.

Dynamic Interconversions between G-Quadruplex Configurations in the Human BCL-2 Proximal Promoter Revealed by Single-Molecule Spectroscopy

B-cell chronic lymphocytic leukemia (CLL) /lymphoma 2 (Bcl-2) gene translates into a mitochondrial membrane protein that inhibits the cell apoptosis and has been identified as a proto-oncogene of varies human cancers. Upstream of the promotor 1 region of Bcl-2, there exists a 39 base-pair GC-rich sequence that potent to fold into G-quadruplex (G4), which potentially regulates the promotor function and becomes a promising cancer-therapeutic drug target by stabilizing G4 structures. However, this sequence carries high polymorphism due to its 6 G-tracts that possibly fold into up to 15 G4 configurations. We utilized single-molecule fluorescence energy transfer microscopy together with circular dichroism spectroscopy to investigate the structural dynamics between G4 configurations. Three co-existing G4 configurations were identified. Among them, a rapid interconversion between two configurations was observed in real-time. We found that the 6th G-tract, which was usually truncated in previous studies, plays a crucial role in mediating the interconversion, presumably, by stabilizing the intermediate. This interconversion also gets retarded in the presence of the G4 stabilizer, pyridostatin (PDS), suggesting a possible common stabilizer chemical can be used as a drug candidate.

Defects in assembly explain reduced antiviral activity of the G249D polymorphism in human TRIM5α.

TRIM5α is an interferon inducible restriction factor which contributes to intrinsic defense against HIV infection by targeting the HIV capsid protein CA. Although human TRIM5α (huTRIM5α) does not potently inhibit HIV-1 infection, the ability of huTRIM5α to exhibit some control of HIV-1 infection is evidenced by a single nucleotide polymorphism in huTRIM5α which substitutes aspartic acid to glycine at position 249 (G249D) in the L2 region and is associated with higher susceptibility to HIV-1 infection. To understand the mechanistic basis for the reduced antiviral activity, we employed biophysical and cell biological methods coupled with molecular dynamics simulations to compare WT and the G249D polymorphism of huTRIM5α. We investigated the differences in conformational dynamics of rhesus and huTRIM5α Coiled Coil–Linker 2 (CC-L2) dimers utilizing circular dichroism and single molecule-Fluorescence Energy Transfer (sm-FRET). These methods revealed that the G249D dimer exhibits secondary structure and conformational dynamics similar to WT huTRIM5α. Homology modelling revealed that G249 was present on the hairpin of the antiparallel dimer, in a position which may act to stabilize the adjacent BBox2 domain which mediates the inter-dimeric contacts required for the formation of TRIM5 assemblies. We therefore asked if the G249D mutant forms assemblies in cells with the same efficiency as WT protein by expressing these proteins as YFP fusions and quantifying the number of assemblies in cells. In cells expressing comparable amounts of protein, the G249D mutant formed fewer assemblies than WT protein, in agreement with our homology modeling predictions and molecular dynamics simulations of dimers and higher oligomers of TRIM5α, providing a mechanistic explanation of the reduced antiviral activity of the G249D polymorphism.

Single-Molecule FRET Methods to Study Glutamate Receptors.

Single-molecule fluorescence energy transfer methods allow us to determine the complete structural landscape between the donor and acceptor fluorophores introduced on the protein of interest. This method is particularly attractive to study ion channel proteins as single-molecule current recordings have been used to study the function of these proteins for several decades. Here we describe the smFRET method used to study glutamate receptors.

New in Vitro Assay Measuring Direct Interaction of Nonsense Suppressors with the Eukaryotic Protein Synthesis Machinery.

Nonsense suppressors (NonSups) induce "readthrough", i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.

Lighting up riboswitching

RNA Folding RNAs fold as they are synthesized, and this folding is required for function. Uhm et al. describe a single-molecule fluorescence energy transfer assay to monitor cotranscriptional RNA folding. This approach revealed folding in the thiamine pyrophosphate (TPP) riboswitch that regulates translation of genes involved in the synthesis of thiamine, an essential vitamin. The riboswitch folds into the “off” conformation, in which translation is inhibited, even in the absence of the TPP ligand. If TPP is not bound to this off conformation, it can switch to the “on” conformation when transcription pauses near the translation start codon, and this allows translation to start. TPP binding stabilizes the off conformation and prevents the switch. The assay will allow investigation of other cases in which transcriptional speed and pausing affect RNA folding. Proc. Natl. Acad. Sci. U.S.A. 10.1073/pnas.1712983115 (2017).

Probing Differential Binding Mechanisms of Phenylalanine-Glycine-Rich Nucleoporins by Single-Molecule FRET.

Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex. They are highly dynamic under physiological conditions and studying their interaction with nuclear transport receptors (NTRs) is key to understanding the molecular mechanism of nucleocytoplasmic transport. Distinct conformational features of FG-Nups interacting with diverse NTRs can be detected by multiparameter single-molecule fluorescence energy transfer (smFRET), which is a powerful technique for studying the dynamics and interactions of biomolecules in solution. Here we provide a detailed protocol utilizing smFRET to reveal differential binding mechanisms of FG-Nups to NTRs, with a focus on practical considerations on sample preparation of unglycosylated and glycosylated FG-Nups, site-specific dual-labeling, smFRET measurements, and data analysis.

The initiation, propagation and dynamics of CRISPR-SpyCas9 R-loop complex.

CRISPR-Cas9 system has been widely used for efficient genome editing. Although the structures of Cas9 protein in complex with single-guided RNA (sgRNA) and target DNA have been resolved, the molecular details about the formation of Cas9 endonuclease R-loop structure remain elusive. Here we examine the DNA cleavage activities of Streptococcus pyogenes Cas9 (SpyCas9) and its mutants using various target sequences and study the conformational dynamics of R-loop structure during target binding using single-molecule fluorescence energy transfer (smFRET) technique. Our results show that Cas9–sgRNA complex divides the target DNA into several distinct domains: protospacer adjacent motif, linker, Seed, Middle and Tail. After seed pairing, the Cas9 transiently retains a semi-active conformation and induces the cleavage of either target or non-target strand. smFRET studies demonstrate that an intermediate state exists in prior to the formation of the fully stable R-loop complex. Kinetics analysis of this new intermediate state indicates that the lifetime of this state increases when the base-pairing length of guide-DNA hybrid duplex increases and reaches the maximum at the size of 18 bp. These data provide new insights into the process of R-loop formation and reveal the source of off-targeting in CRISPR/Cas9 system.

RNA folding retrospective: lessons from ribozymes big and small.

RNA folding retrospective: lessons from ribozymes big and small.

Assembling the Pieces of Protein Puzzles

Dynamic supramolecular assemblies are central to many biological functions, from the processing of genetic information to sensing and signaling. Structural and functional studies face enormous challenges associated with large molecular sizes, transient interactions, extensive motions, and partial disorder. To overcome these problems, we combine simulation and theory with experiment. In our coarse-grained simulation approach, we use ensemble refinement techniques that allow us to integrate data from diverse experiments, including X-ray crystallography, small-angle X-ray scattering (SAXS), spin-label distance measurements (EPR), single-molecule fluorescence energy transfer (FRET), paramagnetic relaxation enhancement (PRE), nuclear magnetic resonance (NMR), and single-particle cryo electron microscopy (cryo-EM). The combination of simulation and experiment allows us to build ensemble representations of the dynamic structures of protein supercomplexes and gain insight into their function.

Origin of multiple intersubunit rotations before EF-G-catalyzed ribosomal translocation through the mRNA with a downstream secondary structure

BackgroundA ribosome can translate through both the single-stranded region and duplex region of messenger RNA (mRNA). Recent single molecule fluorescence energy transfer (smFRET) data showed that when the ribosome translates through the mRNA containing a downstream stem loop, the ribosomal complex exhibits multiple fluctuations between the classical non-rotated and hybrid states before undergoing translocation in the presence of elongation factor G and GTP (EF-G.GTP) of high concentration; by contrast, when the ribosome translates through the mRNA lacking the stem loop, the complex samples the hybrid state approximately once before undergoing translocation.ResultsBased on our proposed model, we provide theoretical analyses of the dynamics of the multiple fluctuations before undergoing mRNA translocation, providing quantitative explanations of the smFRET data.ConclusionsThe good quantitative agreement between the theoretical data and the smFRET data supports the model showing that at saturating EF-G.GTP the multiple fluctuations with the mRNA containing the stem loop occur with EF-G still bound to the ribosome, rather than occur after the release of EF-G from the hybrid state and before the re-binding of EF-G; the multiple fluctuations are induced by occurrences of the futile transition during the mRNA translocation, which is induced by the resistance force resulting from the unwinding of the downstream mRNA stem loop to impede the ribosomal translocation; by contrast, with the mRNA lacking the stem loop, no resistance is present to impede the translocation and thus, no futile transition occurs, giving the ribosomal complex sampling the hybrid state once before undergoing translocation.

Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel

The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a 'virtual sorting' single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations. DOI: http://dx.doi.org/10.7554/eLife.01834.001.

Single Molecule Observation of Cyclization of Short DNA Duplex

In the presented work, a single molecule DNA cyclization assay was used to follow the looping kinetics of single DNA 83 bp molecules, utilizing single molecule fluorescence energy transfer (smFRET) technique. The assay was first prepared in a Na+ free condition and the majority of the DNA was in its unlooped form. A sudden Na+ jump was introduced at different concentrations (0.05-1.75M) and finally yielded DNA in its looping state by annealing the complementary single-strand overhangs of the assay. Looping and unlooping rates were obtained from the kinetic measurements. The result shows a positive and negative linear dependence of the Na+ concentration to the looping and unlooping rate, respectively, until they reach a plateau at 500 mM. The plateau persists until about 1M. For concentrations beyond 1M, an immoderate increase in looping rate is noticed while the unlooping rate does so gradually. Above 1M Na+ there is a preference of looping events that is attributed to the increase of the annealing rate of the overhangs rather than increased flexibility, consistent with earlier studies by Ibrahim Cisse et al. (2012).A protein mediated cyclization assay was also used in experiments with HU protein in which a dramatic increase in the looping rate is noticeable. However in high HU concentration, looping is prohibited implying filament formation.

Riboswitch structure and dynamics by smFRET microscopy.

Riboswitches are structured noncoding RNA elements that control the expression of their embedding messenger RNAs by sensing the intracellular concentration of diverse metabolites. As the name suggests, riboswitches are dynamic in nature so that studying their inherent conformational dynamics and ligand-mediated folding is important for understanding their mechanism of action. Single-molecule fluorescence energy transfer (smFRET) microscopy is a powerful and versatile technique for studying the folding pathways and intra- and intermolecular dynamics of biological macromolecules, especially RNA. The ability of smFRET to monitor intramolecular distances and their temporal evolution make it a particularly insightful tool for probing the structure and dynamics of riboswitches. Here, we detail the general steps for using prism-based total internal reflection fluorescence microscopy for smFRET studies of the structure, dynamics, and ligand-binding mechanisms of riboswitches.

Measuring the Pore-Size and Dynamics of the MscL Mechanosensitive Channel using smFRET

Mechanosensitive ion channels are membrane-bound proteins that let solutes (and water) flow in and out of the cell in response to membrane deformation. They are involved in sensory modalities, like touch, sound, fluid balance, and blood pressure. The mechanosensitive channel of large conductance (MscL) is to date the best studied mechanosensitive channel and serves as a model for channel mechanosensitivity. MscL has been crystallized in the closed form, but not in the open form. Some fundamental questions remain to date not fully answered including - what is the diameter of the open channel pore? Previously, several reports described open channel structure of MscL that was obtained by using either EPR or FRET spectroscopy on large ensembles of MscL channels. Although these studies provided good estimates of the open MscL pore, the size of the pore of a fully open channel still remains to be determined. Here we report a study describing both the open and closed states of MscL using single molecule fluorescence energy transfer (smFRET). We measured the distance changes of a number of residues from the three MscL alpha-helices TM1, TM2 and CP, i.e. I25, A27, and M42 on TM1; Y75 and I96 on TM2 and A110 and V120 on CP. Since we measured two or more sites on each alpha helix, rotational tilting angles, as well as the translational movements of the helices could be obtained. From these measurements, a structure of the protein in the open state was determined, using the crystal structure of the closed channel as a reference. From the obtained open structure we could determine the pore size of the fully open channel. To understand the channel mechanosensitivity, we investigated the dynamics of the conformational change of the channel.

Molecular simulations of the fluctuating conformational dynamics of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) do not possess well-defined three-dimensional structures in solution under physiological conditions. We develop all-atom, united-atom, and coarse-grained Langevin dynamics simulations for the IDP α-synuclein that include geometric, attractive hydrophobic, and screened electrostatic interactions and are calibrated to the inter-residue separations measured in recent single-molecule fluorescence energy transfer (smFRET) experiments. We find that α-synuclein is disordered, with conformational statistics that are intermediate between random walk and collapsed globule behavior. An advantage of calibrated molecular simulations over constraint methods is that physical forces act on all residues, not only on residue pairs that are monitored experimentally, and these simulations can be used to study oligomerization and aggregation of multiple α-synuclein proteins that may precede amyloid formation.