Single-molecule FRET Microscopy Research Articles

Pomab a Novel Monoclonal Type-I Antiphospholipid Antibody, Reduces Thrombin Generation By Selectively Binding to the Open Form of Prothrombin

Background. Anti-prothrombin (anti-PT) antibodies are a type of antiphospholipid antibodies frequently found in antiphospholipid syndrome (APS) patients. Previously, we identified two types of anti-PT antibodies, one binding to epitopes in kringle-1 that are hidden in the closed form but available in the open form (Type-I), and another one in which the epitopes are located in fragment-1 but accessible in both forms (Type-II). Our understanding of their functional characteristics remains limited. Goal. To identify Type-I monoclonal antibodies for mechanistic studies. Methods. Type-I anti-prothrombin antibodies were generated by immunizing mice with fragment-1 and producing monoclonal antibodies using hybridoma technology. Binding mechanisms and epitope recognition for one representative Type-I antibody, POmAb, were elucidated using surface plasmon resonance (SPR), single-molecule FRET (smFRET), and cryo-electron microscopy (cryo-EM). Functional evaluations were performed in human plasma and with purified components. In vivo hemostatic activity was assessed in a laser injury model of the cremasteric arteriole followed by intravital microscopy. Results. Type-I antibodies recognizing kringle-1 were obtained from immunization experiments. One of them, POmAb (Prothrombin Open monoclonal Antibody), was selected for in-depth mechanistic and structural studies. SPR studies showed that POmAb binds to the open form of prothrombin with low nanomolar affinity, and binding was impaired by the mutations S91A and Y93A in kringle-1. smFRET provided evidence of conformational selection, where POmAb preferentially selects the open conformation of prothrombin, thereby shifting the equilibrium towards the open form. Cryo-EM studies of the POmAb-prothrombin complex revealed an extended binding interface in kringle-1 which overlaps with the region occupied by the serine protease domain in the closed form, explaining why POmAb binds to the open form. In human plasma, POmAb prolonged the activated partial thromboplastin time and Russel's viper venom time, though the effect was modest. Further investigations using purified proteins and antibody fragments confirmed that POmAb reduces thrombin generation, although it does not completely abolish it, as the inhibition of thrombin generation occurs through a non-competitive mechanism with prothrombinase. In vivo studies corroborated in vitro findings, further proving reduced fibrin accumulation at the site of injury. Conclusions. We discovered POmAb, a Type-I antiphospholipid antibody. We demonstrated that it selectively binds to the open form of prothrombin, resulting in reduced thrombin generation. POmAb represents a novel tool for studying the role of Type-I antibodies in APS and advances the provocative new idea of conformational trapping of prothrombin as a novel class of anticoagulants.

Single-molecule view of coordination in a multi-functional DNA polymerase.

Replication and repair of genomic DNA requires the actions of multiple enzymatic functions that must be coordinated in order to ensure efficient and accurate product formation. Here, we have used single-molecule FRET microscopy to investigate the physical basis of functional coordination in DNA polymerase I (Pol I) from Escherichia coli, a key enzyme involved in lagging-strand replication and base excision repair. Pol I contains active sites for template-directed DNA polymerization and 5' flap processing in separate domains. We show that a DNA substrate can spontaneously transfer between polymerase and 5' nuclease domains during a single encounter with Pol I. Additionally, we show that the flexibly tethered 5' nuclease domain adopts different positions within Pol I-DNA complexes, depending on the nature of the DNA substrate. Our results reveal the structural dynamics that underlie functional coordination in Pol I and are likely relevant to other multi-functional DNA polymerases.

PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments.

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA-damaging agents and is tightly regulated to restrict TLS to damaged DNA. Replication protein A (RPA), the major single-strand DNA (ssDNA) binding protein complex, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single-molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18·RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination and represent, to the best of our knowledge, the first example of ATP-independent translocation of a protein complex along a protein filament.

Probing RNA Helicase Conformational Changes by Single-Molecule FRET Microscopy.

Förster resonance energy transfer (FRET) is a versatile tool to study the conformational dynamics of proteins. Here, we describe the use of confocal and total internal reflection fluorescence (TIRF) microscopy to follow the conformational cycling of DEAD-box helicases on the single molecule level, using the eukaryotic translation initiation factor eIF4A as an illustrative example. Confocal microscopy enables the study of donor-acceptor-labeled molecules in solution, revealing the population of different conformational states present. With TIRF microscopy, surface-immobilized molecules can be imaged as a function of time, revealing sequences of conformational states and the kinetics of conformational changes.

Dissecting Structure, Function and Dynamics of Molecular Machines by Single-Molecule FRET Microscopy: Translation Initiation is Regulated through Modulation of the Conformational Dynamics of the Dead-Box Protein eIF4A

Dissecting Structure, Function and Dynamics of Molecular Machines by Single-Molecule FRET Microscopy: Translation Initiation is Regulated through Modulation of the Conformational Dynamics of the Dead-Box Protein eIF4A

Molecular Crowding Effects on Stability and Kinetics of Trinucleotide Repeat Hairpins

Numerous genetic disorders arise from the propensity of certain repetitive DNA sequences to form intrastrand, non-helical structures, such as hairpins, due to extensive self-complementarity. Within the cellular environment, the formation of these non-helical structures occurs within highly crowded conditions. Using single-molecule FRET microscopy, the effect of molecular crowding on the stability and dynamics of individual DNA hairpins containing the trinucleotide repeat sequences (CAG)N and (CTG)N were explored. These repeat sequences have central mismatches within each repeat unit that reduce the stability of intrastrand contacts between repeat units in the stem, leading to highly dynamic behavior for their hairpins. The structural dynamics for (CAG)N and (CTG)N trinucleotide repeat hairpins were quantified in the presence of molecular crowding agents (polyethylene glycol; PEG) with different sizes. Analysis of the transitions rates between the unstructured and closer conformations shows that an increase in molecular crowding promotes the formation of the hairpins. Strikingly, the crowding agents induce a greater acceleration of hairpin melting and overall destabilization, which contrasts with observations from controls with fully paired DNA hairpins. Under these conditions, the high mismatch content of these repeat hairpins becomes more destabilizing in the presence of PEG. The findings indicate that molecular crowding may confer some protective benefit to genomic DNA by disrupting these deleterious structures at smaller repeat sizes.

G-Quadruplex and Protein Binding by Single-Molecule FRET Microscopy.

G-quadruplex (G4) is a non-canonical nucleic acid structure that arises from the stacking of planar G-tetrads, stabilized by monovalent cations. G4 forming sequences exist throughout the genome and G4 structures are shown to be involved in many processes including DNA replication and gene expression. The single-molecule total internal reflection fluorescence (TIRF) microscopy has been employed to study G4 structure formation and protein binding interactions. Here, we describe methods by which we tested the folding and unfolding of G-quadruplexes structure and studied the dynamics of its interaction with POT1 protein. The methods presented here can be applied to study other putative G4 sequences and potential binding partners.

Single molecule analysis of structural fluctuations in DNA nanostructures.

DNA origami is an excellent tool for building complex artificial nanostructures. Functionalization of these structures provides the possibility of precise organization of matter at the nanoscale. In practice, efforts in this endeavour can be impeded by electrostatic repulsion or other dynamics at the molecular scale, resulting in uncompliant local structures. Using single molecule FRET microscopy combined with coarse-grained Brownian dynamics simulations, we investigated here the local structure around the lid of a DNA origami box, which can be opened by specific DNA keys. We found that FRET signals for the closed box depend on buffer ion concentrations and small changes to the DNA structure design. Simulations provided a view of the global and local structure and showed that the distance between the box wall and lid undergoes fluctuations. These results provide methods to vizualise and improve the local structure of three-dimensional DNA origami assemblies and offer guidance for exercising control over placement of chemical groups and ligands.

Evaluation and optimisation of unnatural amino acid incorporation and bioorthogonal bioconjugation for site-specific fluorescent labelling of proteins expressed in mammalian cells

Many biophysical techniques that are available to study the structure, function and dynamics of cellular constituents require modification of the target molecules. Site-specific labelling of a protein is of particular interest for fluorescence-based single-molecule measurements including single-molecule FRET or super-resolution microscopy. The labelling procedure should be highly specific but minimally invasive to preserve sensitive biomolecules. The modern molecular engineering toolkit provides elegant solutions to achieve the site-specific modification of a protein of interest often necessitating the incorporation of an unnatural amino acid to introduce a unique reactive moiety. The Amber suppression strategy allows the site-specific incorporation of unnatural amino acids into a protein of interest. Recently, this approach has been transferred to the mammalian expression system. Here, we demonstrate how the combination of unnatural amino acid incorporation paired with current bioorthogonal labelling strategies allow the site-specific engineering of fluorescent dyes into proteins produced in the cellular environment of a human cell. We describe in detail which parameters are important to ensure efficient incorporation of unnatural amino acids into a target protein in human expression systems. We furthermore outline purification and bioorthogonal labelling strategies that allow fast protein preparation and labelling of the modified protein. This way, the complete eukaryotic proteome becomes available for single-molecule fluorescence assays.

Single-Molecule Studies of ssDNA-Binding Proteins Exchange.

Single-stranded DNA-binding protein (SSB) is important not only for the protection of single-stranded DNA (ssDNA) but also for the recruitment of other proteins for DNA replication, recombination, and repair. The interaction of SSB with ssDNA is highly dynamic as it exists as an intermediate during cellular processes that unwind dsDNA. It has been proposed that SSB redistributes itself among multiple ssDNA segments, but transient intermediates are difficult to observe in bulk experiments. We can use single-molecule FRET microscopy to observe intermediates of the transfer of a single Escherichia coli SSB from one ssDNA strand to another or exchange of one SSB for another on a single ssDNA in real time. This single-molecule approach can be further applicable to understand relative binding affinities and competitive dynamics for other SSBs and variants across various systems.

Single - Molecule Fluorescence Microscopy of Folding Dynamics of G-Quadruplex DNA

G-quadruplexes are unique secondary DNA structures that have been proposed to play a number of regulatory roles in cell biology [1] and are possible targets for anti-cancer therapies [2]. Here we employed single-molecule FRET microscopy to investigate the folding and underlying conformational dynamics of G-quadruplexes formed by the human telomeric sequence. Our results yield a comprehensive thermodynamic and kinetic description of the folding of G-quadruplexes that we find to proceed through a complex multi-route pathway, involving several intermediate conformational states. This detailed understanding of the folding process of G-quadruplexes allowed us to shed light into their interaction with specific quadruplex-binding ligands, possessing efficient anti-cancer activity. Moreover, our recent experiments in the presence of synthetic polyethylene glycol-based crowders provide further insights into the folding of G-quadruplexes in molecularly crowded environments. These studies provide a detailed picture of conformational dynamics of G-quadruplexes under a broad range of experimental conditions helping to uncover the common mechanistic features of their folding.(1) Rhodes, D.; Lipps, H. J. Nucleic Acids Res.2015, 43, 8627.(2) Balasubramanian, S.; Hurley, L. H.; Neidle, S. Nat Rev Drug Discov2011, 10, 261.

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes.

G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo. The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales. Combining the single-molecule data with molecular dynamics simulations enabled obtaining a structural description of the experimentally observed folded states. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of G4s that proceeds through a complex multi-route pathway, involving several marginally stable conformational states.

Single-Molecule Conformational Dynamics of E. coli DNA Polymerase I

The extraordinary accuracy of DNA replication is due in part to the coordinated action of various enzymatic activities exhibited by many DNA polymerases. Structural studies have revealed that many of these functions are localized in spatially separated protein domains, but what is not known is how these domains are coordinated with respect to each other and the DNA substrate in order to ensure efficient and accurate DNA synthesis. One such polymerase is DNA polymerase I (Pol I) from E. coli, which is composed of the Klenow fragment (KF), containing the 5’-3’ polymerase and 3’-5’ exonuclease activities, and a separate 5’ nuclease domain attached via a flexible linker. Here we have used single-molecule FRET (smFRET) microscopy to determine the position of the 5’ nuclease domain of Pol I relative to the downstream strand of various immobilized model DNA substrates. A high-FRET state was observed for a substrate containing a downstream flap - the natural substrate for the 5’ nuclease activity - suggesting that the 5’ nuclease domain is docked with the downstream strand of the duplex, as expected. Interestingly, a similar high-FRET state was also observed for a gapped substrate, suggesting that the 5’ nuclease is also near the downstream strand during polymerase activity. Together, these observations may explain the increased processivity of Pol I versus KF during DNA synthesis and suggest that the 5’ nuclease domain may also serve to mediate the switch between strand-displacement synthesis and RNA primer removal during Okazaki fragment processing. Conversely, no FRET was observed for a substrate containing a double mismatch at the 3’ end of the primer, indicating that the 5’ nuclease domain is remote from the downstream strand of the substrate during proofreading activity.

Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na+ solutions by single molecule FRET microscopy.

G-quadruplex structures can occur throughout the genome, including at telomeres. They are involved in cellular regulation and are potential drug targets. Human telomeric G-quadruplex structures can fold into a number of different conformations and show large conformational diversity. To elucidate the different G-quadruplex conformations and their dynamics, we investigated telomeric G-quadruplex folding using single molecule FRET microscopy in conditions where it was previously believed to yield low structural heterogeneity. We observed four FRET states in Na+ buffers: an unfolded state and three G-quadruplex related states that can interconvert between each other. Several of these states were almost equally populated at low to medium salt concentrations. These observations appear surprising as previous studies reported primarily one G-quadruplex conformation in Na+ buffers. Our results permit, through the analysis of the dynamics of the different observed states, the identification of a more stable G-quadruplex conformation and two transient G-quadruplex states. Importantly these results offer a unique view into G-quadruplex topological heterogeneity and conformational dynamics.

NMDA Receptor Ion Channel Dynamics in Living Cells by a Novel Single-Molecule Patch-Clamp FRET Microscopy: Revealing the Multiple Conformational States Associated with a Channel at its Electrical Off State

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels. The conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging for probing ion channel conformational changes simultaneously with the electrical single channel recording. We were able to probe single ion-channel-protein conformational changes simultaneously with the electric on-off signals, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level.(1,2) We have probed NMDA (N-Methyl-D-Aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. Based on our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics. Our results shed light on new perspectives of the intrinsic interplay of lipid membrane dynamics, solvation dynamics, and the ion channel functions.Reference:1. Dibyendu Kumar Sasmal, H. Peter Lu, “ Single-Molecule Patch-Clamp FRET Microscopy Studies of NMDA Receptor Ion Channel Dynamics in Living Cells: Revealing the Multiple Conformational States Associated with a Channel at Its Electrical Off State,” J. Am. Chem. Soc., 136, 12998-13005 (2014).2. Suneth P. Rajapaksha, Xuefei Wang, H. Peter Lu, “Suspended Lipid Bilayer for Optical and Electrical measurements of Single Ion Channel Proteins,” Anal. Chem., 85, 8951-8955 (2013).

Computational Prediction of G-Quadruplex Formation

Guanine-rich regions of genomic DNA can spontaneously fold into secondary structures called G-quadruplexes (GQs). Akin to tiny switches, GQs regulate genetic processes through their folding and unfolding. Their interest to basic science, as well as their potential as therapeutic targets for human diseases, has motivated the creation of computational tools for their prediction. Currently, GQ folding predictors are based on results from structural and biochemical studies of GQ formed in single-stranded (ss) DNA. As a result, existing tools perform poorly when applied to the prediction of GQ formation in double-stranded (ds) DNA, the native context within which genomic GQs are found. Here, we present a probabilistic model of GQ formation, which is learned from large-scale human genomic pull-down experiments and applied to the analysis of gene ontological data. Advances in the characterization of GQs in dsDNA have enabled us to integrate results from small-molecule binding assays and single-molecule FRET microscopy into our model. In order to obtain training sets of sequences, we identified nearly 700,000 unique, potential GQs and categorized them according to pull-down experiment outcomes. Model parameters learned from these training sets agree with experimental evidence and, when asked to predict the folding of dsDNA GQ sequences, outperformed existing models of GQ folding. To further explore the model's utility, we screened potential GQ drug targets by selecting high-scoring sequences for gene ontological analysis. Our results indicate that highly scoring sequences are preferentially located near cancer-related genes. This tool can be applied to genomic sequences to locate the most strongly forming GQs, revealing valuable information for the design of GQ-targeting therapies, and represents the next step toward the practical, widespread use of GQs in medicine and technology.

Fast and User-Friendly Single-Molecule FRET Microscopy Software

Single-molecule FRET (smFRET) microscopy is a powerful tool for monitoring the conformational diversity and structural dynamics of individual biomolecules. However, the full data analysis of single molecule FRET movies is often a complex and time-consuming task. Here, we present a fast, robust and user-friendly data analysis platform for smFRET microscopy of immobilized molecules. The presented toolkit can be operated without any prior programming skills and allows different aspects of smFRET data to be analyzed in an explorative and extensible framework. We illustrate its capabilities using data of different double stranded DNA and G-quadruplex structures.

Temperature-cycle microscopy reveals single-molecule conformational heterogeneity

Our previous temperature-cycle study reported FRET transitions between different states on FRET-labeled polyprolines [Yuan et al., PCCP, 2011, 13, 1762]. The conformational origin of such transitions, however, was left open. In this work, we apply temperature-cycle microscopy of single FRET-labeled polyproline and dsDNA molecules and compare their responses to resolve the conformational origin of different FRET states. We observe different steady-state FRET distributions and different temperature-cycle responses in the two samples. Our temperature-cycle results on single molecules resemble the results in steady-state measurements but reveal a dark state which could not be observed otherwise. By comparing the timescales and probabilities of different FRET states in temperature-cycle traces, we assign the conformational heterogeneity reflected by different FRET states to linker dynamics, dye-chain and dye-dye interactions. The dark state and low-FRET state are likely due to dye-dye interactions at short separations.

Single-molecule patch-clamp FRET microscopy studies of NMDA receptor ion channel dynamics in living cells: revealing the multiple conformational states associated with a channel at its electrical off state.

Conformational dynamics plays a critical role in the activation, deactivation, and open–close activities of ion channels in living cells. Such conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy, to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging with single-channel electric current recording. Our approach is particularly capable of resolving ion channel conformational change rate process when the channel is at its electrically off states and before the ion channel is activated, the so-called “silent time” when the electric current signals are at zero or background. We have probed NMDA (N-methyl-d-aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open–close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. On the basis of our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open–close dynamics.

Function and Movement of a DNA Actuator Investigated by Single Molecule Fret Microscopy

DNA is an interesting building block for the construction of active nanoscale devices. Movement in these structures is typically achieved through conformational changes that can be driven by DNA fuel strands or by internal changes in DNA hybridization. In this study we investigate the function and fidelity of a DNA actuator with 11 states using single molecule FRET microscopy. The DNA actuator is a complex molecular DNA device capable of finely controlled actuation through the movement of its two DNA piston arms, see the included figure. The actuator is designed to have 10 actuation steps of less than 1 nm each and to be locked in a desired state by the addition of specific locking strands. Our results show that the DNA actuator can be effectively locked in place in several conformations with the help of well-designed DNA locking strands. However, depending on the sequence of the locking strands, the device can also show spontaneous oscillations between well-defined states. Our results demonstrate that single molecule studies provide a stringent test for the performance of molecular machines made out of DNA.