Single Binding Event Research Articles

Watching Splicing Live

![Figure][1] CREDIT: DIANA KATHARINE HUNT The spliceosome, which excises introns from precursors to messenger RNAs (pre-mRNAs), is a highly dynamic macromolecular machine consisting of five RNAs and scores of both core and accessory proteins. To overcome the difficulties of fully reconstituting the spliceosome from its purified components, Hoskins et al. (p. [1289][2]) followed its assembly in yeast using colocalization single-molecule spectroscopy (CoSMoS) in unfractionated cell extracts. Their kinetic analysis reveals that the spliceosome assembles by first binding U1 small nuclear RNA followed by U2, U4/U6.U5, and then the multiprotein Prp19-complex. No single subcomplex binding event limits the overall assembly rate, and the commitment of the pre-mRNA molecule to splicing progressively increases as the spliceosome assembles. [1]: pending:yes [2]: /lookup/doi/10.1126/science.1198830

Dynamic and Ordered Assembly of Single Spliceosomes

Spliceosome assembly is a highly dynamic and complex process involving the addition of 5 snRNAs and >100 proteins to a pre-mRNA transcript. This process has largely been analyzed by discontinuous assays that provide limited kinetic information. By combining yeast genetics, chemical biology, and a multi-wavelength single molecule microscopy technique (Colocalization Single Molecule Spectroscopy, CoSMoS), we monitored the formation of single spliceosomes. This approach revealed that individual spliceosome subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes. No single subcomplex binding event disproportionately limits the speed of the assembly reaction. While not all pre-mRNAs acquire a functional spliceosome, commitment of pre-mRNAs to splicing increases as assembly progresses. Therefore, spliceosome assembly is not a one-way, unbranched process as some models depict. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in complex environments.

Dynamic force spectroscopy on the binding of monoclonal antibodies and tau peptides

Optical tweezers-assisted dynamic force spectroscopy (DFS) is employed to investigate specific receptor/ligand interactions on the level of single binding events. Here, the specific binding of two anti-human tau monoclonal antibodies (mAbs), HPT-110 and HPT-104, to synthetic tau-peptides with different phosphorylation patterns is analyzed. The specificity of HPT-110 to the tau-peptide containing a phosphorylation at Ser235 and of HPT-104 to the tau-peptide containing a phosphorylation at Thr231 is confirmed. Additionally, our approach allows for a detailed characterization of the unspecific interactions that are observed between HPT-104 and the peptide phosphorylated only at Ser235 and between HPT-110 and the peptide phosphorylated only at Thr231. By analyzing the measured rupture-force distributions it is possible to separate unspecific from specific interactions. Thereby for the latter characteristic parameters like the lifetime of the bond without force τ0, the characteristic length xts and the free energy of activation ΔG are determined. The results are in accordance with conventional ELISA tests but offer a much more refined insight.

Fully integrated monolithic optoelectronic transducer for real-time protein and DNA detection: The NEMOSLAB approach

The development and testing of a portable bioanalytical device which was capable for real-time monitoring of binding assays was demonstrated. The device was based on arrays of nine optoelectronic transducers monolithically integrated on silicon chips. The optocouplers consisted of nine silicon avalanche diodes self-aligned to nine silicon nitride waveguides all converging to a single silicon detector. The waveguides were biofunctionalized by appropriate recognition molecules. Integrated thick polymer microchannels provided the necessary fluidic functions to the chip. A single sided direct contact scheme through a board-to-board receptacle was developed and combined with a portable customized readout and control instrument. Real-time detection of deleterious mutations in BRCA1 gene related to predisposition to hereditary breast/ovarian cancer was performed with the instrument developed using PCR products. Detection was based on waveguided photons elimination through interaction with fluorescently labeled PCR products. Detection of single biomolecular binding events was also demonstrated using nanoparticles as labels. In addition, label-free monitoring of bioreactions in real time was achieved by exploiting wavelength filtering on photonic crystal engineered waveguides. The proposed miniaturized sensing device with proper packaging and accompanied by a portable instrument can find wide application as a platform for reliable and cost effective point-of-care diagnosis.

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1-2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNalpha, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNalpha were titrated into (15)N-labeled SCR1-2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn(11), Arg(13), Ala(22), Arg(28), Ser(32), Arg(36), Lys(41), Lys(57), Tyr(64), Lys(67), Tyr(68), Arg(83), Gly(84), and Arg(89). With regard to IFNalpha, the binding is similar to the CR2-C3d interaction with specific residues being Arg(13), Tyr(16), Arg(28), Ser(42), Lys(48), Lys(50), Tyr(68), Arg(83), Gly(84), and Arg(89). We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K(d) values of 0.13 and 160 microm, whereas the CR2-gp350 and CR2-IFNalpha interactions were characterized as single site binding events with affinities of 0.014 and 0.035 microm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

Kinetic Mechanism for DNA Unwinding by Multiple Molecules of Dda Helicase Aligned on DNA

Helicases catalyze the separation of double-stranded nucleic acids to form single-stranded intermediates. Using transient state kinetic methods, we have determined the kinetic properties of DNA unwinding under conditions that favor a monomeric form of the Dda helicase as well as conditions that allow multiple molecules to function on the same substrate. Multiple helicase molecules can align like a train on the DNA track. The number of base pairs unwound in a single binding event for Dda is increased from approximately 19 bp for the monomeric form to approximately 64 bp when as many as four Dda molecules are aligned on the same substrate, while the kinetic step size (3.2 +/- 0.7 bp) and unwinding rate (242 +/- 25 bp/s) appear to be independent of the number of Dda molecules present on a given substrate. The data support a model in which the helicase molecules bound to the same substrate move along the DNA track independently during DNA unwinding. The observed increase in processivity arises from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. These results are in contrast to previous reports in which multiple Dda molecules on the same track greatly enhanced the rate and amplitude for displacement of protein blocks on the track. Therefore, only when the progress of the lead molecule in the train is impeded by some type of block, such as a protein bound to DNA, do the trailing molecules interact with the lead molecule to overcome the block. The fact that trailing helicase molecules have little impact on the lead molecule in the train during routine DNA unwinding suggests that the trailing molecules are moving at rates similar to that of the lead molecule. This result implicates a step in the translocation mechanism as contributing greatly to the overall rate-limiting step for unwinding of duplex DNA.

Cytoplasmic Dynein is not a Classical Duty Ratio Motor

Cytoplasmic dynein is not a classical duty ratio motorThe mechanical cross bridge cycle of cytoplasmic dynein has often been compared with that of myosin. Cytoplasmic dynein and myosin 5 are both organelle bound motors responsible for transport of their cargo over a long distance. It has also been demonstrated that myosin 5 is a processive motor and that its processivity is regulated by the duty ratio; i.e., the ratio between bound and free state during the cross bridge cycle. As the binding of ATP leads to the dissociation of the motor filament complex a decrease of the ATP concentration results in an increased duty ratio.It was the aim of our study to investigate whether the processivity of cytoplasmic dynein is also governed by the duty ratio. With the optical trap single molecule measurements were carried out in a two bead dumbbell approach. At 100 μM ATP consecutive 8 nm steps up to stall force were observed often resulting in repeated 8nm forward and backward steps at stall force. To our surprise however, at low ATP concentrations dynein underwent only single binding events with an apparent working stroke of 8nm.These results can not be explained by a simple one site model for ATP binding where processivity is governed by the duty ratio. In contrast to myosin, dynein possesses two essential ATP binding sites. At low ATP concentrations we hypothesis that only one of the ATP binding sites is occupied, thereby resulting in a loss of processivity.

Interaction of Bacterial PI-Specific Phospholipase C With Lipid Bilayer Surfaces

PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is a 40 kDa enzyme that catalyzes the cleavage of GPI-anchored proteins and PI. PI-PLC binds to the membrane via the interfacial binding surface, a special region on the rim of the active site pocket. Hydrophobic and electrostatic interactions (via Trp and Lys residues, respectively) are proposed to play a role in membrane binding. To further examine these interactions, recombinant wild-type (WT) and mutant His6-tagged PI-PLCs (W47A, W242A, W47A/W242A and K44A) were expressed in E. coli and purified. Trp emission of WT and mutant enzymes did not undergo a blue shift upon addition of phosphatidylcholine (PC) liposomes, suggesting that PI-PLC does not deeply penetrate the membrane during membrane association. Trp quenching upon association of WT/mutant PI-PLC with bilayers containing brominated lipids (70% egg PC, 30% 6,7-Br2PC) was equal to that observed for pure egg PC, again suggesting no deep membrane penetration of the enzymes. Stopped-flow fluorescence studies showed first-order quenching of Trp fluorescence upon mixing of WT/mutant PI-PLCs with zwitterionic and negatively charged liposomes, consistent with the notion of a single binding event. The calculated dissociation constants revealed much weaker membrane association of W47A/W242A PI-PLC compared to WT enzyme, and ultimately suggest a role for these Trp residues in membrane association. Introduction of a PI substrate into egg PC liposomes resulted in 5-fold stronger association of WT and mutant PI-PLCs with the membrane surface. Lastly, Langmuir isotherms showed a change in the cross-sectional area per molecule of dimyristoyl-PC and dimyristoylphosphatidylglycerol monolayers upon injection of WT PI-PLC into the subphase, implying that PI-PLC association alters the surface packing of the monolayer. Changes in the area per molecule on PI-PLC binding were more substantial above the Tm of these lipids. (Supported by NSERC).

Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase

RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP and the ATPase cycle fuel the work performed by helicases is not completely clear. The hepatitis C virus RNA helicase, NS3, is an important model system for this class of enzymes. NS3 binding to a single-/double-strand RNA or DNA junction leads to ATP-independent melting of the duplex and formation of a complex capable of ATP-dependent unwinding by using a spring-loaded mechanism. We have established an RNA substrate for NS3 that can be unwound in a single sub-step. Our studies are consistent with a model in which a single ATP binding and/or hydrolysis event sets the unwinding spring and phosphate dissociation contributes to release of the spring, thereby driving the power stroke used for unwinding.

Multivalency in the Assembly of Intrinsically Disordered Dynein Intermediate Chain

Dynein light chains are thought to increase binding efficiency of dynein intermediate chain to both dynein heavy chain and dynactin, but their exact role is not clear. Isothermal titration calorimetry and x-ray crystallography reported herein indicate that multivalency effects underlie efficient dynein assembly and regulation. For a ternary complex of a 60-amino acid segment of dynein intermediate chain (IC) bound to two homodimeric dynein light chains Tctex1 and LC8, there is a 50-fold affinity enhancement for the second light chain binding. For a designed IC construct containing two LC8 sites, observed the 1000-fold enhancement reflects a remarkably pure entropic chelate effect of a magnitude commensurate with theoretical predictions. The lower enhancement in wild-type IC is attributed to unfavorable free energy changes associated with incremental interactions of IC with Tctex1. Our results show assembled dynein IC as an elongated, flexible polybivalent duplex, and suggest that polybivalency is an important general mechanism for constructing stable yet reversible and functionally versatile complexes.

Biochemical basis for the essential genetic requirements of RecA and the β-clamp in Pol V activation

In Escherichia coli, it is genetically well established that the beta-clamp and RecA are essential cofactors that endow DNA polymerase (Pol) V with lesion bypass activity. However, the biochemical basis for these requirements is still largely unknown. Because the process of translesion synthesis (TLS) requires that the specialized DNA polymerase synthesize in a single binding event a TLS patch that is long enough to resist external proofreading, it is critical to monitor Pol V burst synthesis. Here, we dissect the distinct roles that RecA and the beta-clamp perform during the Pol V activation process using physiologically relevant long single-stranded template DNA, similar to those used in genetic assays. Our data show that the beta-clamp endows the complex between Pol V and the template DNA with increased stability. Also, the RecA filament formed in cis on the single-stranded DNA produced downstream from the lesion stretches the template DNA to allow smooth elongation of the nascent strand by Pol V. The concurrent action of both cofactors is required for achieving productive TLS events. The present article presents an integrated view of TLS under physiologically relevant conditions in E. coli that may represent a paradigm for lesion bypass in other organisms.

Moving beyond convergence in the pheromone system of the moth

Introduction Male moths can sense and locate conspecific females releasing small amounts of pheromone from more than a mile away [1]. Integral to this behavior is the extraordinary sensitivity of the male moths olfactory apparatus to pheromone molecules. This apparatus involves the transduction of molecular binding events in large populations of olfactory receptor neurons (ORNs) and their convergent projections onto relatively small numbers of neurons in the macro glomerular complex (MGC). Schneider calculated a behavioral threshold of the pheromone system of 200 molecular binding events at the antennae [1]. Recently Angioy et al. have reported that fluctuations of the male moths cardiac rhythm were elicited with a calculated 6 molecular events at the antennae [2]. While it has been demonstrated that ORN are capable of producing spikes after only a single molecular binding event the overall sensitivity of the systems is limited by noise in the from Eighteenth Annual Computational Neuroscience Meeting: CNS*2009 Berlin, Germany. 18–23 July 2009

Single Molecule Kinetics of ENTH Binding to Lipid Membranes

The Epsin N terminal homology (ENTH) domain is a peripheral membrane protein that transiently associates with membranes by binding the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). This protein exemplifies general principals of spatio-temporal control in lipid-protein and protein-protein interactions. For example, ENTH assists clathrin mediated budding by intercalating into the lipid head group's region thus stabilizing non-spherical membrane shapes. We capture the different stages in tubulation of liposomes induced by ENTH binding using cryo transmission electron microscopy.To gain further insight into the interactions of ENTH with model membranes of well controlled compositions we image single ENTH binding events via total internal reflection fluorescence microscopy. Frame rates in excess of 75 exposures per second and subsequent single fluorophore tracking provides insight into details of ENTH binding kinetics at different PIP2 concentrations. The resulting distributions of residence times and step-sizes of the ENTH diffusion on the membrane show significant deviations from simple kinetics and one component diffusion processes.

Tunable immunonanoparticle binding to cancer cells: thermodynamic analysis of targeted drug delivery vehicles

Tumour cells are often associated with altered surface receptor profiles, and these changes can provide a basis for targeted delivery of anti-cancer agents. Functionalizing a colloidal drug delivery vehicle, such as a polymeric nanoparticle, with several targeting ligands has qualitatively been shown to increase the effective affinity of the nanoparticle for its target receptor over the affinity of the free ligand. However, whether this increase results from multiple simultaneous interactions per particle (multivalent binding) or increased configurations for single binding events per particle (monovalent binding) is unclear. A quantitative approach was required to distinguish between these possible mechanisms. In this study, human epidermal growth factor receptor 2 (HER2) overexpressing cancer cells (SKBR-3) were used as the target for anti-HER2 (trastuzumab, Herceptin™) immunonanoparticles. We varied the antibody conjugation density on the immunonanoparticles and measured their cellular binding by a flow cytometric immunoassay. Using this method, we were able to directly assay the targeted cells and quantify immunonanoparticle binding strength, allowing us to better understand whether immunonanoparticles were bound by monovalent or multivalent interactions. The binding data for each formulation were fitted to Langmuir isotherms, and based on the theory presented herein, it was concluded that the system studied behaved in a manner consistent with monovalent binding. Understanding this property of immunonanoparticle binding is useful in drug delivery applications, where manipulating the strength of such interactions is essential to controlling their targeting capacity on both tissue and cellular levels. The models developed here can be used to quantitatively predict binding strength for rational immunonanoparticle design.

Mitosis: Cdh1 Clears the Way for Anaphase Spindle Assembly

The two co-activators of the anaphase-promoting complex/cyclosome (APC/C), Cdc20 and Cdh1, facilitate ubiquitination by the core complex, but their role in conferring substrate specificity has been contentious. A new report reveals that Cdc20-bound APC/C initiates the timely proteolysis of many Cdh1 substrates with the exception of the Aurora kinases. Failure to degrade Aurora kinases results in abnormal anaphase microtubule organisation and premature cytokinesis.

Nonidentifiability of the Source of Intrinsic Noise in Gene Expression from Single-Burst Data

Over the last few years, experimental data on the fluctuations in gene activity between individual cells and within the same cell over time have confirmed that gene expression is a “noisy” process. This variation is in part due to the small number of molecules taking part in some of the key reactions that are involved in gene expression. One of the consequences of this is that protein production often occurs in bursts, each due to a single promoter or transcription factor binding event. Recently, the distribution of the number of proteins produced in such bursts has been experimentally measured, offering a unique opportunity to study the relative importance of different sources of noise in gene expression. Here, we provide a derivation of the theoretical probability distribution of these bursts for a wide variety of different models of gene expression. We show that there is a good fit between our theoretical distribution and that obtained from two different published experimental datasets. We then prove that, irrespective of the details of the model, the burst size distribution is always geometric and hence determined by a single parameter. Many different combinations of the biochemical rates for the constituent reactions of both transcription and translation will therefore lead to the same experimentally observed burst size distribution. It is thus impossible to identify different sources of fluctuations purely from protein burst size data or to use such data to estimate all of the model parameters. We explore methods of inferring these values when additional types of experimental data are available.

In situ Sensing of Single Binding Events by Localized Surface Plasmon Resonance

Single binding events of nanoparticle-labeled DNA strands were detected as stepwise peak shifts in localized surface plasmon resonance by single particle measurement. We confirmed the number of binding events by observing label particles by scanning electron microscopy. Our simulation based on a multiple multipole program showed that the peak shift is dependent on interparticle gap size and binding position. The experimental peak shift distribution was also reproduced by simulation.

Whispering-gallery-mode biosensing: label-free detection down to single molecules

Optical label-free detectors, such as the venerable surface plasmon resonance (SPR) sensor, are generally favored for their ability to obtain quantitative data on intermolecular binding. However, before the recent introduction of resonant microcavities that use whispering gallery mode (WGM) recirculation, sensitivity to single binding events had not materialized. Here we describe the enhancement mechanisms responsible for the extreme sensitivity of the WGM biosensor, review its current implementations and applications, and discuss its future possibilities.

Stochastic Binding of Ca 2+ Ions in the Dyadic Cleft; Continuous versus Random Walk Description of Diffusion

Ca 2+ signaling in the dyadic cleft in ventricular myocytes is fundamentally discrete and stochastic. We study the stochastic binding of single Ca 2+ ions to receptors in the cleft using two different models of diffusion: a stochastic and discrete Random Walk (RW) model, and a deterministic continuous model. We investigate whether the latter model, together with a stochastic receptor model, can reproduce binding events registered in fully stochastic RW simulations. By evaluating the continuous model goodness-of-fit for a large range of parameters, we present evidence that it can. Further, we show that the large fluctuations in binding rate observed at the level of single time-steps are integrated and smoothed at the larger timescale of binding events, which explains the continuous model goodness-of-fit. With these results we demonstrate that the stochasticity and discreteness of the Ca 2+ signaling in the dyadic cleft, determined by single binding events, can be described using a deterministic model of Ca 2+ diffusion together with a stochastic model of the binding events, for a specific range of physiological relevant parameters. Time-consuming RW simulations can thus be avoided. We also present a new analytical model of bimolecular binding probabilities, which we use in the RW simulations and the statistical analysis.

ENSAM: Europium Nanoparticles for Signal Enhancement of Antibody Microarrays on Nanoporous Silicon

To improve the sensitivity of antibody microarray assays, we developed ENSAM (Europium Nanoparticles for Signal enhancement of Antibody Microarrays). ENSAM is based on two nanomaterials. The first is polystyrene nanoparticles incorporated with europium chelate (beta-diketone) and coated with streptavidin. The multiple fluorophores incorporated into each nanoparticle should increase signal obtained from a single binding event. The second nanomaterial is array surfaces of nanoporous silicon, which creates high capacity for antibody adsorption. Two antibody microarray assays were compared: ENSAM and use of streptavidin labeled with a nine-dentate europium chelate. Analyzing biotinylated prostate-specific antigen (PSA) spiked into human female serum, ENSAM yielded a 10-fold signal enhancement compared to the streptavidin-europium chelate. Similarly, we observed around 1 order of magnitude greater sensitivity for the ENSAM assay (limit of detection < or = 0.14 ng/mL, dynamic range > 10(5)) compared to the streptavidin-europium chelate assay (limit of detection < or = 0.7 ng/mL, dynamic range > 10(4)). Analysis of a titration series showed strong linearity of ENSAM ( R2 = 0.99 by linear regression). This work demonstrates the novel utility of nanoparticles with time-resolved fluorescence for signal enhancement of antibody microarrays, requiring as low as 100-200 zmol biotinylated PSA per microarray spot. In addition, proof of principle was shown for analyzing PSA in plasma obtained from patients undergoing clinical PSA-testing.