Single-molecule Insights Research Articles

Fluidification of Entanglements by a DNA Bending Protein

In spite of the nanoscale and single-molecule insights into nucleoid associated proteins (NAPs), their role in modulating the mesoscale viscoelasticity of entangled DNA has been overlooked so far. By combining microrheology and molecular dynamics simulation, we find that the abundant NAP "integration host factor" (IHF) lowers the viscosity of entangled λDNA 20-fold at physiological concentrations and stoichiometries. Our results suggest that IHF may play a previously unappreciated role in resolving DNA entanglements and in turn may be acting as a "genomic fluidizer" for bacterial genomes.

Identification of substrate-specific allosteric fingerprints in matrix metalloprotease-1 at the single molecule level for precision control of function.

MMPs, a family of 23 human enzymes, interact with and degrade many biomolecules. Any drug targeting the active site of MMPs inhibits intended and unintended functions and results in adverse side effects. If we identify substrate-specific allosteric locations (distant from the active site), we may change one role of MMPs without affecting their other functions for fewer side effects. We attached fluorescent dyes at two specific sites on the two domains of MMP1 to measure inter-domain dynamics on four water-insoluble substrates: collagen fibril, fibrin, alpha-synuclein (aSyn) aggregates, and amyloid-beta (AB) aggregates. We calculated the distance between the two locations from the single molecule Forster Resonance Energy Transfer (smFRET) between the two dyes, measured using a Total Internal Reflection Fluorescence (TIRF) microscope. Low FRET (open) MMP1 conformations with well-separated domains are relevant for collagen, fibrin, and aSyn. In contrast, high FRET (closed) conformations apply to AB aggregates. Functionally relevant conformations are present in active MMP1 but significantly absent in catalytically inactive MMP1. We found that each substrate has unique fingerprints (residues) in MMP1 and leads to changes at the catalytic and distant allosteric sites. Screening drugs against substrate-specific allosteric sites predicted substrate-specific drugs for targeting MMP1 based on single molecule insights.

Mammalian embryo development: Towards a single-molecule perspective.

Revealing how pluripotent cells generate distinct cell lineages in the early mammalian embryo is central to uncovering the mechanisms that drive mammalian development and for unlocking the potential of pluripotent cells for regenerative medicine. The objective of my lab is to provide single-molecule insights into nuclear protein complexes that influence the ability of pluripotent cells to differentiate. Although recent studies have characterised the enzymatic activities of some of these protein complexes, little is known about their intra-nuclear dynamics or how they influence spatiotemporal 3D enhancer-promoter relationships. To investigate these dynamics, we have established tools for live-cell 3D tracking of single HaloTag-tagged nuclear proteins and inactive dCas9-labelled genes. We have also developed machine-learning algorithms to infer properties about the resulting trajectories, allowing us to determine the chromatin binding kinetics of key nuclear protein complexes and also how they control chromatin movement at specific enhancers/promoters. Using these approaches, we reveal the chromatin binding kinetics of two major protein complexes that regulate differentiation - the chromatin remodeller NuRD and the histone methyltransferase KMT2B. Furthermore, we show that both NuRD and KMT2B influence the range genes explore within the nucleus. We then use single-cell transcriptomics (scRNAseq) and chromosome conformation capture (Hi-C) to show that these changes in chromatin movement are linked to the length-scale over which enhancers activate transcription at nearby genes. We propose a model linking the movement of enhancers to gene activation as pluripotent cells differentiate. Our results highlight the importance of making dynamic measurements at single-cell and single-molecule resolution to provide insight into transcriptional control during differentiation. We are currently establishing these technologies within live mouse embryos and we believe this will help the field understand the underlying molecular mechanisms of cell-fate decisions in vivo.

Quantitative Single-Molecule Electrochemiluminescence Bioassay.

A single-molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single-molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single-molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single-molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.

A Single-Molecule Insight into the Ionic Strength-dependent, Cationic Peptide Nucleic Acids-Oligonucleotides Interactions.

To alleviate solubility-related shortcomings associated with the use of neutral peptide nucleic acids (PNA), a powerful strategy is incorporate various charged sidechains onto the PNA structure. Here we employ a single-molecule technique and prove that the ionic current blockade signature of free poly(Arg)-PNAs and their corresponding duplexes with target ssDNAs interacting with a single α-hemolysin (α-HL) nanopore is highly ionic strength dependent, with high salt-containing electrolytes facilitating both capture and isolation of such complexes. Our data illustrate the effect of low ionic strength in reducing the effective volume of free poly(Arg)-PNAs and augmentation of their electrophoretic mobility while traversing the nanopore. We found that unlike in high salt electrolytes, the specific hybridization of cationic moiety-containing PNAs with complementary negatively charged ssDNAs in a salt concentration as low as 0.5 M is dramatically impeded. We suggest a scenario in which reduced charge screening by counterions in low salt electrolytes enables non-specific, electrostatic interactions with the anionic backbone of polynucleotides, thus reducing the ability of PNA-DNA complementary association via hydrogen bonding patterns. We applied an experimental strategy with spatially-separated poly(Arg)-PNAs and ssDNAs, and present evidence at the single-molecule level suggestive of the real-time, long-range interactions-driven formation of poly(Arg)-PNA-DNA complexes, as individual strands entering the nanopore from opposite directions collide inside a nanocavity.

Identification of allosteric fingerprints of alpha-synuclein aggregates in matrix metalloprotease-1 and substrate-specific virtual screening with single molecule insights

Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previous reports showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. This paper reports quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is unavailable, binding poses were predicted by molecular docking of MMP1 with aSyn using ClusPro. MMP1 dynamics were simulated using predicted binding poses and compared with the experimental interdomain dynamics to identify an appropriate pose. The selected aSyn-MMP1 binding pose near aSyn residue K45 was simulated and analyzed to define conformational changes at the catalytic site. Allosteric residues in aSyn-bound MMP1 exhibiting strong correlations with the catalytic motif residues were compared with allosteric residues in free MMP1, and aSyn-specific residues were identified. The allosteric residues in aSyn-bound MMP1 are K281, T283, G292, G327, L328, E329, R337, F343, G345, N346, Y348, G353, Q354, D363, Y365, S366, S367, F368, P371, R372, V374, K375, A379, F391, A394, R399, M414, F419, V426, and C466. Shannon entropy was defined to quantify MMP1 dynamics. Virtual screening was performed against a site on selected aSyn-MMP1 binding poses, which showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for potential substrate-specific control of MMP1 activity in the future. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Single-molecule studies of helicases and translocases in prokaryotic genome-maintenance pathways

Helicases involved in genomic maintenance are a class of nucleic-acid dependent ATPases that convert the energy of ATP hydrolysis into physical work to execute irreversible steps in DNA replication, repair, and recombination. Prokaryotic helicases provide simple models to understand broadly conserved molecular mechanisms involved in manipulating nucleic acids during genome maintenance. Our understanding of the catalytic properties, mechanisms of regulation, and roles of prokaryotic helicases in DNA metabolism has been assembled through a combination of genetic, biochemical, and structural methods, further refined by single-molecule approaches. Together, these investigations have constructed a framework for understanding the mechanisms that maintain genomic integrity in cells. This review discusses recent single-molecule insights into molecular mechanisms of prokaryotic helicases and translocases.

Single-molecule insight into stalled replication fork rescue in Escherichia coli.

DNA replication forks stall at least once per cell cycle in Escherichia coli. DNA replication must be restarted if the cell is to survive. Restart is a multi-step process requiring the sequential action of several proteins whose actions are dictated by the nature of the impediment to fork progression. When fork progress is impeded, the sequential actions of SSB, RecG and the RuvABC complex are required for rescue. In contrast, when a template discontinuity results in the forked DNA breaking apart, the actions of the RecBCD pathway enzymes are required to resurrect the fork so that replication can resume. In this review, we focus primarily on the significant insight gained from single-molecule studies of individual proteins, protein complexes, and also, partially reconstituted regression and RecBCD pathways. This insight is related to the bulk-phase biochemical data to provide a comprehensive review of each protein or protein complex as it relates to stalled DNA replication fork rescue.

Single-Molecule Insights into Allosteric Regulation of the Nuclear Receptor Protein RXRα

Nuclear receptors are crucial transcription factors that respond to a broad range of external signaling ligands by binding cognate DNA response elements and switching on transcription. As such, these proteins form tightly regulated, multi-component allosteric systems. RXRα is one of the key players in this protein family for its role as a promiscuous heterodimerization partner. We employ single-molecule fluorescence techniques to investigate the conformational dynamics of RXRα's ligand binding domain (LBD)—the essential regulatory domain. It has been proposed that ligand binding changes the conformation of helix 12 of the LBD and positions it in a conformation favoring coactivator peptide binding. Our single-molecule FRET studies, however, demonstrate that helix 12 undergoes a substantial conformational change only after the coactivator peptide binds. This suggests an unexpected allosteric mechanism of nuclear receptor activation, which we investigate. In particular, we address the questions: on what time scales do the conformational changes of helix 12 take place? and how are its dynamics affected by modulations in other parts of RXRα and in its binding partners? Our studies will pave the way to better understanding of the complex regulatory machinery embodied in nuclear receptor function.

Combining SPR with atomic-force microscopy enables single-molecule insights into activation and suppression of the complement cascade

Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·μm−2. AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.

Single-molecule insights into the temperature and pressure dependent conformational dynamics of nucleic acids in the presence of crowders and osmolytes

In this review we discuss results from temperature and pressure dependent single-molecule Förster resonance energy transfer (smFRET) studies on nucleic acids in the presence of macromolecular crowders and organic osmolytes. As representative examples, we have chosen fragments of both DNAs and RNAs, i.e., a synthetic DNA hairpin, a human telomeric G-quadruplex and the microROSE RNA hairpin. To mimic the effects of intracellular components, our studies include the macromolecular crowding agent Ficoll, a copolymer of sucrose and epichlorohydrin, and the organic osmolytes trimethylamine N-oxide, urea and glycine as well as natural occurring osmolyte mixtures from deep sea organisms. Furthermore, the impact of mutations in an RNA sequence on the conformational dynamics is examined. Different from proteins, the effects of the osmolytes and crowding agents seem to strongly dependent on the structure and chemical make-up of the nucleic acid.

Single-Molecule Insights into the Temperature Dependent Conformational Changes of a RNA Thermometer in the Presence of Crowders and Osmolytes

Single-Molecule Insights into the Temperature Dependent Conformational Changes of a RNA Thermometer in the Presence of Crowders and Osmolytes

Single-molecule insights into surface-mediated homochirality in hierarchical peptide assembly

Homochirality is very important in the formation of advanced biological structures, but the origin and evolution mechanisms of homochiral biological structures in complex hierarchical process is not clear at the single-molecule level. Here we demonstrate the single-molecule investigation of biological homochirality in the hierarchical peptide assembly, regarding symmetry break, chirality amplification, and chirality transmission. We find that homochirality can be triggered by the chirality unbalance of two adsorption configuration monomers. Co-assembly between these two adsorption configuration monomers is very critical for the formation of homochiral assemblies. The site-specific recognition is responsible for the subsequent homochirality amplification and transmission in their hierarchical assembly. These single-molecule insights open up inspired thoughts for understanding biological homochirality and have general implications for designing and fabricating artificial biomimetic hierarchical chiral materials.

Single-Molecule Insight into the Activation of Human Dynein by Adapter Proteins

Dynactin and cargo adapter proteins dramatically increase the processive motility of human cytoplasmic dynein towards the microtubule minus end, but until now their effect on dynein's force generation remained unknown. Using an optical trap, we confirm that dynein alone is a weak motor and show that dynein's association with dynactin and BICD2N substantially increases its stall force. We propose that activated dynein complexes are strong motors that generate forces comparable to those of plus end-directed kinesins.

Single-molecule insight into Wurtz reactions on metal surfaces.

Wurtz reactions feature the dehalogenated coupling of alkyl halides. In comparison to their widely investigated counterparts, Ullmann reactions, Wurtz reactions have however been scarcely explored on surfaces. Herein, by combining high-resolution STM imaging and DFT calculations, we have systematically investigated Wurtz reactions on three chemically different metal surfaces including Cu(110), Ag(110) and Au(111). We find that the Wurtz reactions could be achieved on all three surfaces, and the temperatures for triggering the reactions are in the order of Cu(110) > Ag(110) > Au(111). Moreover, DFT calculations have been performed to unravel the pathways of on-surface Wurtz reactions and identify three basic steps of the reactions including debromination, diffusion and coupling processes. Interestingly, we found that the mechanism of the on-surface Wurtz reaction is intrinsically different from the Ullmann reaction and it is revealed that the coupling process is the rate-limiting step of Wurtz reactions on three different substrates. These findings have given a comprehensive picture of Wurtz reactions on metal surfaces and demonstrated that such a reaction could be an alternative reaction scheme for advanced on-surface synthesis.

Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

Single-molecule insights into mRNA dynamics in neurons.

Targeting of mRNAs to neuronal dendrites and axons plays an integral role in intracellular signaling, development, and synaptic plasticity. Single-molecule imaging of mRNAs in neurons and brain tissue has led to enhanced understanding of mRNA dynamics. Here we discuss aspects of mRNA regulation as revealed by single-molecule detection, which has led to quantitative analyses of mRNA diversity, localization, transport, and translation. These exciting new discoveries propel our understanding of the life of an mRNA in a neuron and how its activity is regulated at the single-molecule level.

Single-Molecule Insights on Human RNA Polymerase II Transcription Regulation: Assembly of the Pre-Initiation Complex and the Roles of Activators

Transcription of all protein-coding genes in human cells begins with the assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) composed of more than forty polypeptides (total size of ∼ 3MDa). Due to the high complexity and the dynamic nature of the PIC, the mechanism of its assembly and regulation remains elusive after decades of conventional biochemical studies.We have developed a surface-based, promoter-specific Pol II transcription system suitable for single-molecule imaging (Genes and Development 2012, 26:1691). Counting of transcripts at each DNA template suggested a stochastic initiation mechanism in the experimental system. We found a prototypical activator (human Sp1) regulates transcription by enhancing PIC assembly (presumably by recruiting TFIID). Real-time TFIID binding to DNA was monitored and coupled to transcription detection at the same DNA template for the first time. We also developed methods to detect the production of RNA transcripts in real-time and couple that to the kinetic measurements of RNA polymerase binding at the single-molecule level. using multiple fluorescently labeled General Transcription Factors (GTFs, namely TFIIB TFIID, TFIIE, TFIIF and TFIIH) and Pol II, we are currently investigating the structure of PIC, pathways of its assembly, and the mechanism of transcription modulation by sequence-specific activators and the core promoter DNA elements.

There and back again: new single-molecule insights in the motion of DNA repair proteins

Cellular DNA repair machines are constantly at work supporting the integrity of our genomes. Numerous proteins cooperate to form a complex and adaptive system dedicated to detection and timely processing of DNA damage. The molecular underpinnings of how these proteins locate and discriminate DNA lesions, match homologous sequences, mend the DNA and attend to a replication in distress are of a paramount biomedical importance, but in many cases remain unclear. Combined with more conventional tools, single-molecule biochemistry has been stepping in to address the age-old problems in the DNA repair field. This review will address new insights into diffusive properties of three DNA repair systems: I will discuss the emerging model of how MutS homologues locate and respond to mismatches in the dsDNA; the mechanism by which RAD52 promotes annealing of complementary DNA strands coated with ssDNA binding protein RPA; and how the nucleoprotein filament formed by RecA recombinase on ssDNA searches for homology within duplex DNA. These three distinct DNA repair factors exemplify the dynamic nature of cellular DNA repair machines revealed by single-molecule studies.

Scanning Tunneling Microscopy Reveals Single-Molecule Insights into the Self-Assembly of Amyloid Fibrils

Many severe diseases are associated with amyloid fibril deposits in the body caused by protein misfolding. Structural information on amyloid fibrils is accumulating rapidly, but little is known about the assembly of peptides into fibrils at the level of individual molecules. Here we investigate self-assembly of the fibril-forming tetrapeptides KFFE and KVVE on a gold surface under ultraclean vacuum conditions using scanning tunneling microscopy. Combined with restrained molecular dynamics modeling, we identify peptide arrangements with interesting similarities to fibril structures. By resolving individual peptide residues and revealing conformational heterogeneities and dynamics, we demonstrate how conformational correlations may be involved in cooperative fibril growth. Most interestingly, intermolecular interactions prevail over intramolecular interactions, and assembly of the phenyl-rich KFFE peptide appears not to be dominated by π-π interactions. This study offers interesting perspectives for obtaining fundamental single-molecule insights into fibril formation using a surface science approach to study idealized model systems.