Single-molecule Fluorescence Studies Research Articles

Heterogeneous Diffusion in Thin Polymer Films As Observed by High-Temperature Single-Molecule Fluorescence Microscopy

Single-molecule fluorescence microscopy was used to investigate the dynamics of perylene diimide (PDI) molecules in thin supported polystyrene (PS) films at temperatures up to 135 °C. Such high temperatures, so far unreached in single-molecule spectroscopy studies, were achieved using a custom-built setup which allows for restricting the heated mass to a minimum. This enables temperature-dependent single-molecule fluorescence studies of structural dynamics in the temperature range most relevant to the processing and to applications of thermoplastic materials. In order to ensure that polymer chains were relaxed, a molecular weight of 3000 g/mol, clearly below the entanglement length of PS, was chosen. We found significant heterogeneities in the motion of single PDI probe molecules near T(g). An analysis of the track radius of the recorded single-probe molecule tracks allowed for a distinction between mobile and immobile molecules. Up to the glass transition temperature in bulk, T(g,bulk), probe molecules were immobile; at temperatures higher than T(g,bulk) + 40 K, all probe molecules were mobile. In the range between 0 and 40 K above T(g,bulk) the fraction of mobile probe molecules strongly depends on film thickness. In 30-nm thin films mobility is observed at lower temperatures than in thick films. The fractions of mobile probe molecules were compared and rationalized using Monte Carlo random walk simulations. Results of these simulations indicate that the observed heterogeneities can be explained by a model which assumes a T(g) profile and an increased probability of probe molecules remaining at the surface, both effects caused by a density profile with decreasing polymer density at the polymer-air interface.

Effects of DNA Methylation on the Structure of Nucleosomes

Nucleosomes are the fundamental packing units of the eukaryotic genome. Understanding the dynamic structure of a nucleosome is a key to the elucidation of genome packaging in eukaryotes, which is tied to the mechanisms of gene regulation. CpG methylation of DNA is an epigenetic modification associated with the inactivation of transcription and the formation of a repressive chromatin structure. Unraveling the changes in the structure of nucleosomes upon CpG methylation is an essential step toward the understanding of the mechanisms of gene repression and silencing by CpG methylation. Here we report single-molecule and ensemble fluorescence studies showing how the structure of a nucleosome is affected by CpG methylation. The results indicate that CpG methylation induces tighter wrapping of DNA around the histone core accompanied by a topology change. These findings suggest that changes in the physical properties of nucleosomes induced upon CpG methylation may contribute directly to the formation of a repressive chromatin structure.

Role of Solvation Effects in Protein Denaturation: From Thermodynamics to Single Molecules and Back

Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy still surrounds the underlying mechanism by which these molecules denature proteins. Here we review current thinking on various aspects of chemical denaturation. We begin by discussing classic models of protein folding and how the effects of denaturants may fit into this picture through their modulation of the collapse, or coil-globule transition, which typically precedes folding. Subsequently, we examine recent molecular dynamics simulations that have shed new light on the possible microscopic origins of the solvation effects brought on by denaturants. It seems likely that both denaturants operate by facilitating solvation of hydrophobic regions of proteins. Finally, we present recent single-molecule fluorescence studies of denatured proteins, the analysis of which corroborates the role of denaturants in shifting the equilibrium of the coil-globule transition.

Single-Molecule Fluorescence Study on Membrane Proteins Derived from Living Organisms: Application to Drosophila Olfactory Receptor Or83b

Or83b is a broadly expressed receptor protein for Drosophila melanogaster olfaction, but its exact function has not yet been identified. It is still sharply debated whether Or83b is a simple signal mediator in cooperation with other receptors or Or83b itself acts as a part of a cation channel. Here, we describe a general methodology for investigating the function of membrane proteins, derived from living organisms, using single-molecule fluorescence techniques. We reconstituted Or83b and other membrane proteins from Drosophila antennae in 50-nm sized vesicles.

Maximizing Photon Counts and Time Resolution for Single Molecule Fluorescence Studies in Micro Fluidic Mixing System

Since the late 1990s microfabricated diffusive mixers have found applications in a growing number of chemical and biochemical kinetic assays. Microfabricated diffusive mixers require only microliters of sample, and use laminar flow to decouple laboratory time from reaction progress. These attributes make them ideally suited for kinetic measurements with single-molecule sensitivity. The time resolution is set by the time required to diffusively mix and the dwell time needed to insure a given number of photons.

Dynamic Competition for the Proximal RNA Binding Site of Hfq Drives Efficient Annealing of Small Noncoding RNA and mRNA

Hfq, a bacterial RNA chaperone, stimulates specific RNA-RNA interactions by inducing RNA unwinding or RNA-RNA annealing. However, it is still unclear how Hfq can selectively achieve two completely opposite functions--annealing, and unwinding--depending on RNA substrates. Also, basic properties like an oligomeric state of an active form is under debate. Here we describe single-molecule fluorescence studies on Hfq-mediated annealing of DsrA, a small noncoding regulatory RNA of E. coli, and its mRNA target rpoS, which encodes the σStranscription factor. Our results reveal that a single hexamer of Hfq stimulates DsrA-rpoS annealing by simultaneously binding both RNAs using same RNA binding site on the proximal surface of Hfq. The competition for the same binding site of the two RNAs makes the RNA-Hfq interaction dynamic, but drives more efficient annealing. When the Hfq-binding sequence exists only in one of the two RNAs, RNA unwinding can occur due to the reduced stability of the RNA caused by the partial unwinding of the RNA upon Hfq binding on the specific binding site.

Zero-Mode Waveguides: A Powerful Tool for Single-Molecule Optical Studies

Single-molecule fluorescence studies of enzymes that incorporate fluorescently labeled substrate nucleotides typically operate at substrate concentrations well below their Km values. While this is inevitable in conventional fluorescence microscopy, the biological relevance of the insights gained into enzyme mechanism may be compromised. Zero-mode waveguides (ZMWs) provide an excellent solution to this problem by greatly reducing the observation volume. We report the nanofabrication of ZMWs, the surface treatment for controlled immobilization of biomolecules and the reduction of background noise. We also present the development of an assay to monitor in real time the incorporation of fluorescently-labeled nucleotides, which paves the way for the studies of nucleic acid polymerizing enzymes, e.g. DNA/RNA polymerase, reverse transcriptase, telomerase, etc.

Potential of protoporphyrin IX and metal derivatives for single molecule fluorescence studies

Metalloporphyrins are cofactors of a variety of proteins, and are often used as spectroscopic probes of the active site. Many high resolution techniques, such as single molecule spectroscopy, are based on fluorescence contrast and require the replacement of the native metalloporphyrin by a fluorescent analog. We have investigated the potential of several fluorescent analogs of heme, namely free-base protoporphyrin IX and its metal derivatives containing Zn, Sn, and Mg, for single molecule fluorescence studies by determining their room-temperature molecular absorption cross sections and fluorescence quantum yields. According to these data, free-base protoporphyrin IX and its Zn derivative, which have the highest fluorescence quantum yields, are the most suitable heme analogs for single molecule fluorescence studies.

Structure and assembly of bacteriophage T4 head

The bacteriophage T4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. The past twenty years of research has greatly elevated the understanding of phage T4 head assembly and DNA packaging. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as that found in phage HK97 and several other icosahedral bacteriophages. Folding of gp23 requires the assistance of two chaperones, the E. coli chaperone GroEL and the phage coded gp23-specific chaperone, gp31. The capsid also contains two non-essential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. The structure of Soc shows two capsid binding sites which, through binding to adjacent gp23 subunits, reinforce the capsid structure. Hoc and Soc have been extensively used in bipartite peptide display libraries and to display pathogen antigens including those from HIV, Neisseria meningitides, Bacillus anthracis, and FMDV. The structure of Ip1*, one of the components of the core, has been determined, which provided insights on how IPs protect T4 genome against the E. coli nucleases that degrade hydroxymethylated and glycosylated T4 DNA. Extensive mutagenesis combined with the atomic structures of the DNA packaging/terminase proteins gp16 and gp17 elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. Cryo-EM structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at a rate of up to 2000 bp/sec, the fastest reported to date of any packaging motor. FRET-FCS studies indicate that the DNA gets compressed during the translocation process. The current evidence suggests a mechanism in which electrostatic forces generated by ATP hydrolysis drive the DNA translocation by alternating the motor between tensed and relaxed states.

Effect of Single-Strand Break on Branch Migration and Folding Dynamics of Holliday Junctions

The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states.

Acidification of the Oxygen Scavenging System in Single-Molecule Fluorescence Studies: In Situ Sensing with a Ratiometric Dual-Emission Probe

For most of the single-molecule fluorescence studies to date, biomolecules of interest are labeled with small organic dyes which suffer from their limited photostability evidenced by blinking and photobleaching. An enzymatic oxygen scavenging system of glucose oxidase and catalase is widely used to improve the dye photostability but with the unfavorable side effect of producing gluconic acid. It is known that accumulation of this byproduct in solution can lead to considerable acidification, but the uncertainty in its severity under experimentally relevant conditions has been a long-standing area of concern due to the lack of a suitable assay. In this paper we report a fluorescence-based analytical assay for quantitatively assessing the acidification of oxygen scavenging systems in situ. By using a ratiometric, dual-emission dye, SNARF-1, we observed the presence and, for the first time, measured the severity of solution acidification due to the oxygen scavenging system for a number of conditions relevant to single-molecule studies. On the basis of the quantitative analysis of the acidification profile under these conditions, practical guidelines for optimizing the oxygen scavenging system are provided. This in situ assay should be applicable to a large variety of future single-molecule fluorescence studies.

Simultaneous Single-Molecule Fluorescence and Conductivity Studies Reveal Distinct Classes of Aβ Species on Lipid Bilayers

The extracellular senile plaques prevalent in brain tissue in Alzheimer's disease (AD) are composed of amyloid fibrils formed by the Abeta peptide. These fibrils have been traditionally believed to be featured in neurotoxicity; however, numerous recent studies provide evidence that cytotoxicity in AD may be associated with low-molecular weight oligomers of Abeta that associate with neuronal membranes and may lead to membrane permeabilization and disruption of the ion balance in the cell. The underlying mechanism leading to disruption of the membrane is the subject of many recent studies. Here we report the application of single-molecule optical detection, using fluorescently labeled human Alphabeta40, combined with membrane conductivity measurements, to monitor the interaction of single-oligomeric peptide structures with model planar black lipid membranes (BLMs). In a qualitative study, we show that the binding of Alphabeta to the membrane can be described by three distinctly different behaviors, depending on the Alphabeta monomer concentration. For concentrations much below 10 nM, there is uniform binding of monomers over the surface of the membrane with no evidence of oligomer formation or membrane permeabilization. Between 10 nM and a few hundred nanomolar, the uniform monomer binding is accompanied by the presence of peptide species ranging from dimers to small oligomers. The dimers are not found to permeabilize the membrane, but the larger oligomers lead to permeabilization with individual oligomers producing ion conductances of <10 pS/pore. At higher concentrations, perhaps beyond physiologically relevant concentrations, larger extended and dynamic structures are found with large conductances (hundreds of picosiemens), suggesting a major disruption of the membrane.

Single Molecule Analysis of the Mitotic Kinesin Eg5

In vertebrates, proper formation of the mitotic spindle requires the activity of Eg5, a motor protein of the kinesin-5 family. Loss of Eg5 function leads to monopolar spindles and mitotic arrest. Eg5's homotetrameric configuration, in which two pairs of motor domains are connected by a central stalk, allows it to crosslink and slide microtubules. Previous single molecule fluorescence studies of Eg5 have shown that in physiological ionic strength its motion along single microtubules is predominantly diffusive and ATP-independent, but becomes predominantly directional upon crosslinking two microtubules. The structural and mechanistic basis of this allosteric regulation of Eg5's motor activity is poorly understood. One possibility is that Eg5's non-motor domains, including the central stalk and C-terminal tail, transmit signals of microtubule binding state across the Eg5 tetramer and regulate motor activity. To probe the roles of Eg5's non-motor domains and to establish structure-function relationships for these domains, we have generated a series of GFP-labeled Eg5 deletion constructs, including a dimeric version and tetrameric versions containing deletions of the motor domain and of the C-terminal tail domain. Each of the constructs, including one that lacks the kinesin motor domain, binds microtubules with varying affinities. In a multiple motor microtubule gliding assay, deletion of the tail domain results in a 50% increase in gliding velocity, from 15 nm/s for full-length Eg5, to 23 nm/s for tail-less Eg5. This indicates that the tail domain is capable of attenuating Eg5 motility. To understand the contributions of the Eg5's domains to its directional and diffusive movement, we are using single molecule fluorescence microscopy to characterize the motility along microtubules for each construct. These studies suggest a model wherein the non-motor domains of Eg5 modulate its motor activity and contribute to its ability to associate with microtubules.

Chapter 12 - A Highly Purified, Fluorescently Labeled In Vitro Translation System for Single-Molecule Studies of Protein Synthesis

Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful tool for mechanistic investigations of increasingly complex biochemical systems. Recently, we and others have successfully used smFRET to directly investigate the role of structural dynamics in the function and regulation of the cellular protein synthesis machinery. A significant challenge to these experiments, and to analogous experiments in similarly complex cellular machineries, is the need for specific and efficient fluorescent labeling of the biochemical system at locations that are both mechanistically informative and minimally perturbative to the biological activity. Here, we describe the development of a highly purified, fluorescently labeled in vitro translation system that we have successfully designed for smFRET studies of protein synthesis. The general approaches we outline should be amenable to single-molecule fluorescence studies of other complex biochemical systems.

Tetrameric Chimera DK4mer is a Tool to Study Mechanisms Of Kinesin-5 Regulation. A Tetrameric Chimera of a Kinesin 1 and a Kinesin 5 is a Fast Microtubule Sliding Motor

The homo-tetrameric motor protein Eg5 from X. laevis drives relative sliding of anti-parallel microtubules by the processive action of its two opposing sets of dimeric motor. As shown by Kwok et al. (2006, Nat. Chem. Biol. 2:480) and Kapitein et al. (2008, J. Cell Biol. 182:421), tetrameric motors move slowly (∼20nm/s), but processively on a single microtubule alternating between diffusional and directional episodes, while motors moving between two microtubules move in a highly directional and processive fashion.In order to obtain a tetrameric model system with more clearly defined properties and motile phases, we have constructed a tetrameric chimera by replacing Eg5-motor domain and neck-linker by the homologous regions of D. melanogaster Kinesin 1 (DK4mer).In surface-gliding assays, Dk4mer showed fast motility (553±31nm/s), irrespective of a C-terminal his- or GFP-his-tag. Comparison to DmKHC shows a similar k0.5, ATP of ∼0.06mM, suggesting that the GFP-tagged version is suitable for single-molecule fluorescence studies. Single GFP-tagged DK4mer motors moved processively along the MT at speeds comparable to those seen in surface-gliding assays (499±3nm/s). We observe clearly distinguished directional and diffusional episodes and an overall run length of ∼9μm on average. We further performed relative sliding assays using DK4mer and observe the expected trimodal distribution of velocities at v=0, v=v1 and v=2v1, v1 being 500nm/s, clearly showing that DK4mer is capable of sliding microtubules apart simultaneously using both pairs of motor domains.The DK4mer is thus an excellent model system to study regulatory aspects of Kinesin-5 due to its high speed, its long processivity and its clear separation of diffusive and directional motility and its fast and efficient relative sliding of microtubules.(∗authors contributed equally)

Single-molecule fluorescence studies of RNA: A decade’s progress

Over the past decade, single-molecule fluorescence studies have elucidated the structure–function relationship of RNA molecules. The real-time observation of individual RNAs by single-molecule fluorescence has unveiled the dynamic behavior of complex RNA systems in unprecedented detail, revealing the presence of transient intermediate states and their kinetic pathways. This review provides an overview of how single-molecule fluorescence has been used to explore the dynamics of RNA folding and catalysis.

Single-Molecule Electrocatalysis by Single-Walled Carbon Nanotubes

We report a single-molecule fluorescence study of electrocatalysis by single-walled carbon nanotubes (SWNTs) at single-reaction resolution. Applying super-resolution optical imaging, we find that the electrocatalysis occurs at discrete, nanometer-dimension sites on SWNTs. Single-molecule kinetic analysis leads to an electrocatalytic mechanism, allowing quantification of the reactivity and heterogeneity of individual reactive sites. Combined with conductivity measurements, this approach will be powerful to interrogate how the electronic structure of SWNTs affects the electrocatalytic interfacial charge transfer, a process fundamental to photoelectrochemical cells.

Single-molecule Visualization of Binding Modes of Helicase to DNA on PEGylated Surfaces

Abstract Binding modes of helicase to various DNA substrates were visualized by single-molecule fluorescence imaging on silicate coverslips coated with poly(ethylene glycol) (PEG) which minimizes the background noise from nonspecific protein adsorption. The results clearly show that the helicase has higher affinity for double-stranded DNA (dsDNA) with a single-stranded DNA (ssDNA) tail than that without one and that multiple helicases can bind to 4.7-kilonucleotide (knt) ssDNA. The study reported here will enhance the capabilities of single-molecule fluorescence studies on DNA–protein interactions.

Direct Observation of NS3 Substeps at Single Base Pair Resolution

Helicases are a ubiquitous class of nucleic acid motor proteins. They utilize ATP to catalyze the opening of double-stranded nucleic acids. Previous crystallographic and single molecule fluorescence studies have implied that a single ATP molecule would be used for every base pair (bp) being opened by a helicase. However, this has not been directly observed. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. Previous single molecule optical tweezers studies on NS3 unwinding have revealed an 11-bp periodicity with 3.6 bp substeps on average. By building an optical tweezers instrument with high spatial resolution and low drift, we were able to monitor the discrete unwinding substeps of NS3 on RNA at single base pair resolution. We show that under conditions where ATP binding limits the motor dwell time between successive substeps, single base pair substeps are clearly visible. We present a distribution of these substep sizes and the implication of this result on mechanisms of NS3 catalyzed RNA base pair opening and strand release. The distribution of motor dwell times between substeps gave us a direct measure of the coupling ratio between ATP and base pair opening, and we propose an integrated model to rationalize all these observations.

Zero-Mode Waveguides for Real-Time Observation of Single Nucleotide Incorporation

Single-molecule fluorescence studies on the incorporation of fluorescently labeled nucleotides by DNA polymerizing enzymes typically operate at nucleotide concentrations well below their Km values. While this is inevitable given the femtoliter observation volumes accessed via conventional fluorescence microscopy, the biological relevance of the insights gained into enzymatic kinetics may be compromised. Zero-mode waveguides (ZMWs), sub-wavelength holes in a thin metal film, provide an excellent solution to this problem by greatly reducing the observation volume [M. J. Levene, et al., 2003, Science 299, 682-686].We have successfully designed and fabricated ZMWs of about 100 nm in width. In addition we have developed a surface treatment protocol based on PEG functionalization to make the ZMWs biocompatible, and to facilitate the controlled tethering of biomolecules [A. Crut, et al., Nanotechnology, in press (2008)]. In these structures, we have observed the real-time observation of single nucleotide incorporation at biologically-relevant concentrations in ZMWs, The latest scientific results will be presented.