Single-molecule Fluorescence Resonance Energy Research Articles

Thermodynamic compensation to temperature extremes in B. subtilis vs T. maritima lysine riboswitches

T. maritima and B. subtilis are bacteria that inhabit significantly different thermal environments, ∼80 vs. ∼40°C, yet employ similar lysine riboswitches to aid in the transcriptional regulation of the genes involved in the synthesis and transport of amino acids. Despite notable differences in G-C basepair frequency and primary sequence, the aptamer moieties of each riboswitch have striking similarities in tertiary structure, with several conserved motifs and long-range interactions. To explore genetic adaptation in extreme thermal environments, we compare the kinetic and thermodynamic behaviors in T. maritima and B. subtilis lysine riboswitches via single-molecule fluorescence resonance energy transfer analysis. Kinetic studies reveal that riboswitch folding rates increase with lysine concentration while the unfolding rates are independent of lysine. This indicates that both riboswitches bind lysine through an induced-fit (“bind-then-fold”) mechanism, with lysine binding necessarily preceding conformational changes. Temperature-dependent van’t Hoff studies reveal qualitative similarities in the thermodynamic landscapes for both riboswitches in which progression from the open, lysine-unbound state to both transition states (‡) and closed, lysine-bound conformations is enthalpically favored yet entropically penalized, with comparisons of enthalpic and entropic contributions extrapolated to a common [K+] = 100 mM in quantitative agreement. Finally, temperature-dependent Eyring analysis reveals the TMA and BSU riboswitches to have remarkably similar folding/unfolding rate constants when extrapolated to their respective (40 and 80°C) environmental temperatures. Such behavior suggests a shared strategy for ligand binding and aptamer conformational change in the two riboswitches, based on thermodynamic adaptations in number of G-C basepairs and/or modifications in tertiary structure that stabilize the ligand-unbound conformation to achieve biocompetence under both hyperthermophilic and mesothermophilic conditions.

Integration of smFRET and molecular dynamics to characterize conformational dynamics of the spike protein of wild-type SARS-CoV-2 and its variants.

Since the rise of the COVID-19 pandemic, researchers have pushed for development of efficient therapeutics and vaccines to assist in preventing the severity of its viral effects. A major target has been the trimeric SARS-CoV-2 spike protein. There has been various experimental and computational efforts to characterize the structural dynamics of this protein at different stages of its mechanisms. Among these methods are single molecule fluorescence resonance energy transfer (smFRET) experiments as well as molecular dynamics (MD) simulations. The purpose of our current work here is to combine the existing data from smFRET experiments and MD simulations to provide a more complete picture of prefusion SARS-CoV-2 spike protein conformational dynamics. We have particularly examined the conformational dynamics of several variants of this protein in a comparative manner. Specifically, we have examined multiple computational methods based on “implicit screening” approach within different approximations to predict dye-dye distance and FRET efficiency distributions to provide reliable interpretations for both smFRET experimental data and MD simulation data of various spike protein variants including the wild-type, the alpha, beta, gamma, delta, and omega variants as well as the engineered spike protein used in the Moderna vaccine. This work sheds light on the structural heterogeneity of the spike protein in different variants of SARS-CoV-2.

Different Forms of Disorder in NMDA-Sensitive Glutamate Receptor Cytoplasmic Domains Are Associated with Differences in Condensate Formation.

The N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor (NMDAR) helps assemble downstream signaling pathways through protein interactions within the postsynaptic density (PSD), which are mediated by its intracellular C-terminal domain (CTD). The most abundant NMDAR subunits in the brain are GluN2A and GluN2B, which are associated with a developmental switch in NMDAR composition. Previously, we used single molecule fluorescence resonance energy transfer (smFRET) to show that the GluN2B CTD contained an intrinsically disordered region with slow, hop-like conformational dynamics. The CTD from GluN2B also undergoes liquid-liquid phase separation (LLPS) with synaptic proteins. Here, we extend these observations to the GluN2A CTD. Sequence analysis showed that both subunits contain a form of intrinsic disorder classified as weak polyampholytes. However, only GluN2B contained matched patterning of arginine and aromatic residues, which are linked to LLPS. To examine the conformational distribution, we used discrete molecular dynamics (DMD), which revealed that GluN2A favors extended disordered states containing secondary structures while GluN2B favors disordered globular states. In contrast to GluN2B, smFRET measurements found that GluN2A lacked slow conformational dynamics. Thus, simulation and experiments found differences in the form of disorder. To understand how this affects protein interactions, we compared the ability of these two NMDAR isoforms to undergo LLPS. We found that GluN2B readily formed condensates with PSD-95 and SynGAP, while GluN2A failed to support LLPS and instead showed a propensity for colloidal aggregation. That GluN2A fails to support this same condensate formation suggests a developmental switch in LLPS propensity.

Dynamically probing ATP-dependent RNA helicase A-assisted RNA structure conversion using single molecule fluorescence resonance energy transfer.

RNA helicase A (RHA) as a member of DExH-box subgroup of helicase superfamily II, participates in diverse biological processes involved in RNA metabolism in organisms, and these RNA-mediated biological processes rely on RNA structure conversion. However, how RHA regulate the RNA structure conversion was still unknown. In order to unveil the mechanism of RNA structure conversion mediated by RHA, single molecule fluorescence resonance energy transfer was adopted to in our assay, and substrates RNA were from internal ribosome entry site of foot-and-mouth disease virus genome. We first found that the RNA structure conversion by RHA against thermodynamic equilibrium in vitro, and the process of dsRNA YZ converted to dsRNA XY through a tripartite intermediate state. In addition, the rate of the RNA structure conversion and the distribution of dsRNA YZ and XY were affected by ATP concentrations. Our study provides real-time insight into ATP-dependent RHA-assisted RNA structure conversion at the single molecule level, the mechanism displayed by RHA may help in understand how RHA contributes to many biological functions, and the basic mechanistic features illustrated in our work also underlay more complex protein-assisted RNA structure conversions.

Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.

Fret Study of the Binding of HIV-1 RRE RNA to Rev Peptide

In the viral life cycle of HIV-1, binding of the Rev protein to the Rev Response Element (RRE) RNA to form the Rev-RRE complex is essential for viral RNA to be exported from the cell nucleus. This interaction has been suggested as a new therapeutic target, which makes an accurate and detailed understanding of Rev-RRE interactions essential. The m6A post-transcriptional modification is known to regulate gene expression and modulate infectivity of viral RNAs. The binding region of the RRE RNA has two known, conserved m6A methylation sites, but the effect and significance of these modifications is an active area of investigation. This study measures binding of an Alexa Fluor 594-labeled RNA hairpin that contains the high-affinity Rev-binding site to an Alexa Fluor 488-labeled peptide that contains the 17 amino acid binding region of the Rev protein as a model of the primary RRE-Rev binding site. Binding of Rev peptides to immobilized RNA hairpins is measured via single molecule fluorescence resonance energy transfer (FRET) spectroscopy. Single molecule measurements can access information hidden by averaging in bulk measurements, especially heterogeneities, such as multiple conformations, short lived intermediates, or varied kinetic rates. Statistical analysis and a kinetic model enable extraction of binding and dissociation rates for the Rev protein. The eventual goal of this study is a comparison of the binding rates for methylated and unmethylated variants of the RRE RNA, which will reveal whether m6A methylation significantly impacts the Rev-RRE binding rate at the primary binding site.

Ligand-bound glutamine binding protein assumes multiple metastable binding sites with different binding affinities

Protein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.

Conformational spread and dynamics in allostery of NMDA receptors

Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer-dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.

An Integrative Approach to Single-Molecule FRET Spectroscopy and Molecular Dynamics Simulations for the Study of Intrinsically Disordered Proteins

Recent technological advances in single-molecule techniques, such as single molecule fluorescence resonance energy transfer (smFRET) spectroscopy, have paved the way for the study of protein structural dynamics under biologically relevant conditions. Unlike high-resolution structure determination techniques such as x-ray crystallography, however, smFRET spectroscopy provides limited information spatially. Fortunately, the smFRET data combined with computational techniques such as molecular dynamics (MD) simulations could provide enough information to model the structural dynamics of protein at a spatiotemporal resolution. The starting point of these simulations is often based on crystal structure or other high-resolution structures of proteins. Unfortunately, such structures are not available for intrinsically disordered proteins (IDPs). Here we have employed enhanced sampling techniques in combination with all-atom MD and smFRET data to model the structural dynamics of IDPs. We have specifically employed state-of-the-art dimensionality reduction techniques in conjunction with enhanced sampling schemes to assimilate MD trajectories to make them consistent with the smFRET measurements. The novel methodology developed in this work uses the smFRET data within enhanced sampling techniques to first generate an ensemble of protein conformations and then predict FRET efficiency distributions for candidate labeled proteins. An iterative procedure is then used to efficiently design smFRET experiments based on the computational predictions and refine the conformational ensemble of protein, until a convergence is reached. The final step involves employing machine learning techniques to determine both the state distributions and their kinetics from smFRET-informed MD trajectories. We have successfully used this approach to study the structural dynamics of the C-terminal domain of membrane insertase Alb3, which is known to be intrinsically disordered. The novel approach developed here lays the groundwork for the efficient application of smFRET technique for the study of IDPs.

Characterizing the impact of splicing protein Dib1 on the pre‐messenger RNA interactions with the spliceosome

In eukaryotic organisms, pre‐messenger RNA splicing facilitates the removal of non‐coding regions of RNA to create a functional mRNA. Pre‐mRNA splicing is catalyzed by the spliceosome, a large, dynamic, molecular machine composed of five small nuclear ribonucleoproteins (snRNPs). Dib1, a key protein of the spliceosome, resides in the U4/U6.U5 triple‐snRNP. Dib1 is essential for cell viability and is conserved from yeast to humans. Dib1 must depart the splicing complex before the catalytic steps of splicing can occur; however, how Dib1's departure is facilitated is unknown. Dib1 and pre‐mRNA are in close proximity in the pre‐catalytic spliceosome (Plaschka et al. Nature 2017); characterizing the interactions between Dib1 and pre‐mRNA may be necessary to understand Dib1's departure. We are characterizing potential interactions between Dib1 and pre‐mRNA using single molecule fluorescence colocalization experiments in collaboration with Dr. Nils Walter at the University of Michigan. An open question is how and whether Dib1 and pre‐mRNA are co‐localized in the splicing machinery. We are addressing this question using Cy5‐labeled Dib1 and Cy3‐labeled pre‐mRNA. We have labeled purified Dib1 with a Cy5 fluorophore and have been able to splice Cy3‐labeled pre‐mRNA. Progress and results of the single molecule fluorescence colocalization between Dib1 and pre‐mRNA will be discussed. Another area of interest is how temperature‐sensitive mutant Dib1, which causes a loss of splicing function in Saccharomyces cerevisiae, may affect the pre‐mRNA throughout the splicing cycle. To this end, we are characterizing pre‐mRNA conformation in S. cerevisiae extracts containing wild type or mutant Dib1 protein. We are using single molecule fluorescence resonance energy transfer (smFRET) to analyze the conformation of pre‐mRNA labeled with a Cy3 fluorophore at the 5′ splice site and a Cy5 fluorophore at the branch point. Comparison of the smFRET analysis will show whether mutant Dib1 causing loss of function is associated with a change of RNA conformation. Progress and results of the smFRET analysis of pre‐mRNA will be discussed.Support or Funding InformationNIH R15GM120720; Robert A Welch Foundation Grant W‐1905This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.

Direct observation of multiple conformational states in Cytochrome P450 oxidoreductase and their modulation by membrane environment and ionic strength

Cytochrome P450 oxidoreductase (POR) is the primary electron donor in eukaryotic cytochrome P450 (CYP) containing systems. A wealth of ensemble biophysical studies of Cytochrome P450 oxidoreductase (POR) has reported a binary model of the conformational equilibrium directing its catalytic efficiency and biomolecular recognition. In this study, full length POR from the crop plant Sorghum bicolor was site-specifically labeled with Cy3 (donor) and Cy5 (acceptor) fluorophores and reconstituted in nanodiscs. Our single molecule fluorescence resonance energy transfer (smFRET) burst analyses of POR allowed the direct observation and quantification of at least three dominant conformational sub-populations, their distribution and occupancies. Moreover, the state occupancies were remodeled significantly by ionic strength and the nature of reconstitution environment, i.e. phospholipid bilayers (nanodiscs) composed of different lipid head group charges vs. detergent micelles. The existence of conformational heterogeneity in POR may mediate selective activation of multiple downstream electron acceptors and association in complexes in the ER membrane.

Transient intermediates are populated in the folding pathways of single-domain two-state folding protein L.

Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.

Single molecule FRET investigation of pressure-driven unfolding of cold shock protein A.

We demonstrate that fused silica capillaries are suitable for single molecule fluorescence resonance energy transfer (smFRET) measurements at high pressure with an optical quality comparable to the measurement on microscope coverslips. Therefore, we optimized the imaging conditions in a standard square fused silica capillary with an adapted arrangement and evaluated the performance by imaging the focal volume, fluorescence correlation spectroscopy benchmarks, and FRET measurements. We demonstrate single molecule FRET measurements of cold shock protein A unfolding at a pressure up to 2000 bars and show that the unfolded state exhibits an expansion almost independent of pressure.

Revealing the Mechanism of Amyloid Fibril Formation by Combined Single Molecule FRET and Kinetic Modeling

The conversion of native soluble proteins into insoluble amyloid fibrils that are rich in cross β-sheet structure is associated with a variety of neurodegenerative diseases. It has recently become apparent that smaller oligomers formed during the early stages of amyloid fibril formation may be primarily responsible for the toxicity in amyloid diseases. Traditional ensemble techniques have limitations in characterization of oligomers due to the metastable and heterogeneous nature of these prefibrillar species. Single molecule fluorescence spectroscopy allows the conformational dynamics of individual biomolecules to be investigated, revealing properties that may be averaged in the ensemble experiment. It is therefore extremely powerful for identification and characterization of the low-populated, heterogeneous and transient species formed during fibril assembly. Combining single molecule fluorescence with kinetic modeling can provide quantitative information regarding the microscopic steps in amyloid formation. Here we have applied single molecule fluorescence resonance energy transfer to investigate in detail the intermolecular assembly and aggregation process of the yeast prion protein Ure2. Oligomerization during the initial lag phase was observed, and two types of Ure2 oligomers with different assembly modes were identified. Furthermore, using theoretical analysis combined with single molecule and ensemble kinetic data, we describe the formation and depletion pathway of oligomers, and propose a multi-step mechanism for Ure2 fibril formation, in which initial oligomerization is followed by conformational conversion to β-sheet containing oligomers that are then able to grow to form mature amyloid fibrils. We also show directly that secondary nuclei do not form, and that fragmentation is therefore responsible for the autocatalytic behavior of Ure2, in contrast to the mechanism of the amyloid formation of Aβ peptide. These results shed light on the potential origin of differences in cytotoxicity between functional and disease-related amyloids.

Structural dynamics of Giα protein revealed by single molecule FRET

The heterotrimeric G proteins (Gαβγ) act as molecular switches to mediate signal transduction from G protein-coupled receptors to downstream effectors. Upon interaction with an activated receptor, G protein exchanges its bound GDP with GTP, stimulating downstream signal transmission. Release of GDP requires a structural rearrangement between the GTPase domain and helical domain of the Gα subunit. Here, we used single molecule fluorescence resonance energy transfer (smFRET) technique to study the conformational dynamics of these two domains in the apo state and in the binding of different ligands. Direct imaging of individual molecules showed that the Giα subunit is highly dynamic, and at least three major conformations of Giα could be observed in the apo state. Upon binding of GDP, Giα becomes dramatically less dynamic, resulting in a closed conformation between the two domains. We postulate that changes between the three conformations are sequential, and the three conformations appear to have distinct affinities toward GDP.

Single Molecules in Focus: From RNA Splicing to Silencing

Nature and Nanotechnology likewise employ nanoscale machines that self-assemble into structures of complex architecture and functionality. Fluorescence microscopy offers a non-invasive tool to probe and ultimately dissect and control these nanoassemblies in real-time. In particular, single molecule fluorescence resonance energy transfer (smFRET) allows us to measure distances at the 2-8 nm scale, whereas complementary super-resolution localization techniques based on Gaussian fitting of imaged point spread functions (PSFs) measure distances in the 10 nm and longer range. First, I will describe how we used smFRET to show that single spliceosomes - responsible for the accurate removal of all intervening sequences (introns) in pre-m(essenger) RNAs - are working as biased Brownian ratchet machines. Second, we have developed Single Molecule Cluster Analysis (SiMCAn) based on a large smFRET dataset to further dissect the complex conformational dynamics of the pre-mRNA as it is spliced. Third, I will describe a method for the intracellular single molecule, high-resolution localization and counting (iSHiRLoC) of microRNAs (miRNAs), a large group of gene silencers with profound roles in our body, from stem cell development to cancer. Microinjected, singly-fluorophore labeled, functional miRNAs were tracked at super-resolution within individual diffusing particles. Observed mobility and mRNA dependent assembly changes suggest the existence of two kinetically distinct assembly processes. We are currently feeding these data into a single molecule systems biology pipeline to bring into focus the unifying molecular mechanism of such a ubiquitous gene regulatory pathway.

Effect of Phosphorylation on Supertertiary Structure and Ligand Binding Affinity on Scaffold Proteins

PSD-95 and PSD-93 are multidomain scaffold proteins with a similar tertiary structure and a common set of interaction partners. Despite the high sequence homology in their protein binding domains these isoforms play opposing roles in learning and memory. The origin of their functional differences remains a mystery. The most apparent physical difference between them is the length and composition of their interdomain linkers as well as the number and position of the phosphorylation sites. In PSD-95, the binding domains partition into two structurally-independently supramodules separated by an intrinsically disordered linker. PSD-93 has a similar intrinsically disordered region but the supertertiary structure has yet to be determined. We used single molecule fluorescence resonance energy transfer (FRET) to compare the supertertiary structures and found PSD-93 has a greater separation between supramodules. This could arise from the increased linker length but also a more even distribution of positive and negative charges in PSD-93. The number and spatial organization of charges determines the extension of intrinsically-disordered peptides. Interestingly, in vitro phosphorylation by Src kinase decreased the separation in PSD-93 but increased the separation in PSD-95 resulting a similar separation between supramodules upon phosphorylation. As with structural studies, binding between PSD-95 and its ligands has been extensively studied while the affinity of PSD-93 for their shared ligands is has not been measured. Using smFRET we found that the two scaffolds have similar binding affinity, before and after phosphorylation, for their common ligands. Furthermore, we found that phosphorylation selectively increased the affinity for some ligands while leaving others unchanged. Our results show that phosphorylation can remodel the supertertiary structure of multidomain scaffold proteins. Additionally, phosphorylation can change the specificity by selectively altering binding affinity for specific ligands.

Expanding the Scope of Single Molecule FRET Spectroscopy towards Primarily Undergraduate Institutions

Single molecule fluorescence methods provide a unique and ultrasensitive set of probes for monitoring the conformations and dynamics of protein chains within physiologically relevant contexts. In particular, single molecule fluorescence resonance energy transfer (smFRET) is capable of monitoring the distances between two fluorescent dyes with sub-nanometer spatial and nanosecond temporal resolution. Unfortunately, smFRET remains inaccessible to many biophysical researchers and most of all to students at primarily undergraduate research institutions. This is in part due to the difficulties involved in making dual-labelled protein samples but also stems from the expense and/or complexity of existing single-molecule fluorescence detection platforms. To address the first issue, we have developed a sample generation protocol suitable for making large libraries of dual-labelled proteins and ribosome-bound nascent chains (RNCs). This method consists of using a purified and reconstituted in-vitro translation system for protein expression, the incorporation of azide or alkyne-bearing non-standard amino acids, and finally click-chemistry-based dye attachment. Here, we show that this approach is simple and robust enough to be used by undergraduate students to make large libraries of dual-labelled protein samples in about a day. To enable smFRET-based screening of these libraries we have also built the first-ever confocal smFRET microscope at a primarily undergraduate institution. This instrument was built for less than $20K largely using second-hand optical components. It has a 3-axis piezo-scanning stage which enables single-molecule imaging and trajectory analysis on surface-immobilized molecules. It also has alternating laser excitation capabilities which enable digital sorting of single molecules using multi-dimensional histograms. This work marks the first implementation of smFRET as a research tool at a primarily undergraduate research institution.

Study of Bloom resolving G-quadruplex process by using high resolution magnetic tweezer with illumination of total internal reflection

G-quadruplex (G4) is a DNA structure which commonly exists in human genome, and it is considered as an important structure in DNA metabolism such as replication, transcription and homologous recombination. The G-quadruplex helicases have been widely investigated these years. Of them, the Bloom (BLM) helicase is most thoroughly studied. However, there are some basic problems that are still unclear. Most of previous studies of G4 are performed by single molecule fluorescence resonance energy transfer technique. The G4 is in a free state in these experiments, which is different from the physiological environment in cells. The traditional magnetic tweezers have a limitation of spatial resolution in a low force circumstance. Thus here we use high resolution magnetic tweezer under the illumination of total internal reflection fluorescence to study the process of BLM resolving G4. Our modification of magnetic tweezer is to separate the measurements of force and distance of magnetic tweezer in order to improve the spatial resolution, which allows us to observe the unfolding process of G4. With a 2-3 pN force we find that the process of BLM unfolding G4 in low ATP concentration is stepwise, and the G4 is mainly in the state between G-quadruplex and G-triplex. We also find that the BLM could interact with G4 for a long time. Our apparatus is also able to obtain the long time observation results compared with the single molecule fluorescence technique. So we perform experiments with a nearly saturated ATP concentration. We find that the BLM has two ways to maintain G4 dissolution in this condition. The BLM could unfold the G4 repetitively in a long period and it could also keep the G4 in unfolding state for a long time after it has opened the G4. Finally, we also perform single molecule fluorescence resonance energy transfer experiment in the same condition, and we find that the 2-3 pN force in magnetic tweezers has a rare influence on the process of BLM interacting with G4. The results of single molecule fluorescence resonance energy transfer experiments are corresponding to the results of magnetic tweezer in the same conditions. All of our experimental results show that ATP dependent BLM has a high affinity with G4 and BLM has a different way to resolve G4 in high ATP concentration. These results could provide new ideas of the mechanism of BLM resolving G4. Our modified magnetic tweezer shows its capacity in G4 single molecule study, and it could be a useful tool in the future single molecule studies.