Single Molecule Binding Assays Research Articles

Ionic strength alters crosslinker-driven self-organization of microtubules.

The microtubule cytoskeleton is a major structural element inside cells that directs self-organization using microtubule-associated proteins and motors. It has been shown that finite-sized, spindle-like microtubule organizations, called "tactoids," can form in vitro spontaneously from mixtures of tubulin and the antiparallel crosslinker, MAP65, from the MAP65/PRC1/Ase family. Here, we probe the ability of MAP65 to form tactoids as a function of the ionic strength of the buffer to attempt to break the electrostatic interactions binding MAP65 to microtubules and inter-MAP65 binding. We observe that, with increasing monovalent salts, the organizations change from finite tactoids to unbounded length bundles, yet the MAP65 binding and crosslinking appear to stay intact. We further explore the effects of ionic strength on the dissociation constant of MAP65 using both microtubule pelleting and single-molecule binding assays. We find that salt can reduce the binding, yet salt never negates it. Instead, we believe that the salt is affecting the ability of the MAP65 to form phase-separated droplets, which cause the nucleation and growth of tactoids, as recently demonstrated.

Inhibition of NUPR1-Karyopherin β1 Binding Increases Anticancer Drug Sensitivity.

Background: Nuclear protein-1 (NUPR1, also known as p8/Com-1) is a transcription factor involved in the regulation of cellular stress responses, including serum starvation and drug stimulation. Methods: We investigated the mechanism of NUPR1 nuclear translocation involving karyopherin β1 (KPNB1), using a single-molecule binding assay and confocal microscopy. The cellular effects associated with NUPR1–KPNB1 inhibition were investigated by gene expression profiling and cell cycle analysis. Results: The single-molecule binding assay revealed that KPNB1 bound to NUPR1 with a binding affinity of 0.75 nM and that this binding was blocked by the aminothiazole ATZ-502. Following doxorubicin-only treatment, NUPR1 was translocated to the nucleus in more than 90% and NUPR1 translocation was blocked by the ATZ-502 combination treatment in MDA-MB-231 with no change in NUPR1 expression, providing strong evidence that NUPR1 nuclear translocation was directly inhibited by the ATZ-502 treatment. Inhibition of KPNB1 and NUPR1 binding was associated with a synergistic anticancer effect (up to 19.6-fold) in various cancer cell lines. NUPR1-related genes were also downregulated following the doxorubicin–ATZ-502 combination treatment. Conclusion: Our current findings clearly demonstrate that NUPR1 translocation into the nucleus requires karyopherin β1 binding. Inhibition of the KPNB1 and NUPR1 interaction may constitute a new cancer therapeutic approach that can increase the drug efficacy while reducing the side effects.

Mapping Transmembrane Binding Partners for E-Cadherin Ectodomains

Transmembrane proteins play essential roles in coupling cells and in sensing mechanical and biochemical signals from the environment. However, it is extremely challenging to identify membrane protein interactions using traditional methods like affinity pulldown. While proximity-tagging methods have been employed to map membrane protein interactomes, these assays merely report on protein proximity and testing direct interactions requires use of a complementary biophysical tool. Here, we combine proximity labeling and single molecule binding assays, to discover novel transmembrane protein interactions in cells.

Mapping transmembrane binding partners for E-cadherin ectodomains

We combine proximity labeling and single molecule binding assays to discover transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a "bait" protein with BioID biotin ligase and identify proximal proteins that are biotin tagged on both their extracellular and intracellular regions. We then test direct binding interactions between proximal proteins and the bait, using single molecule atomic force microscope binding assays. Using this approach, we identify binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-α2β1, the receptor tyrosine kinase ligand ephrin-B1, and the classical cadherin P-cadherin, all directly interact with E-cadherin ectodomains. Our data shows that combining extracellular and cytoplasmic proximal tagging with a biophysical binding assay increases the precision with which transmembrane ectodomain interactors can be identified.

Abstract 5138: Inhibition of kPNB1 and NUPR1 binding increase the anti-cancer drug sensitivity

Abstract Karyopherin beta 1 (KPNB1, known as importin beta 1) is carrier protein, it is known as transporter of protein from cytoplasm to nucleus through nuclear pore complexes (NPCs) using KPNAs as an adapters. The nuclear protein-1 (NUPR1, known as p8/Com-1) is identified as a transcription factor and it is showed multifunctional mechanisms related to cellular stress response such as serum-starvation, oxidative stress and drug stimulation. In this study, we investigated protein-protein interaction between NUPR1 and KPNB1, it might be a new target for cancer therapy.For the evaluation of binding affinity between protein and inhibition effect by compound 1, we processed the single molecule binding assay. Cytotoxicity of combination treatment with doxorubicin and compound 1 were validated several cancer cell lines such as MDA-MB-231, PC-3, SK-OV-3, and SiHa. Gene expression patterns were analyzed RNA-Seq and validated with qRT-PCR and western-blotting. KPNB1 bind to NUPR1 as a high affinity compare to KPNB1 single treatment assay and there was no difference according to concentration of NUPR1. KPNB1 and NUPR1 protein-protein interaction was inhibited by compound 1 according to concentration of compound 1 showed 71%, 44%, and 31% of inhibition rate with 50nM, 10nM, 2.5nM of compound 1, respectively. In doxorubicin single treatment, NUPR1 move to nucleus more than 90% of cells compare to non-treated MDA-MB-231 cells. Otherwise, NUPR1 was exist both nucleus and cytoplasm more than 90% cells, which is indicate that translocation of NUPR1 to the nucleus was blocked by compound 1 treatment. And combination treatment showed synergistic effect of cytotoxicity in various cancer cells such as such as MDA-MB-231, SiHa, PC-3, and SK-OV-3 (1.4 ~ 6 fold). Additionally, NUPR1 related genes were showed change of gene expression in compound 1 combination compared to doxorubicin single treatment. In these results suggest that translocation mechanism of NUPR1 into nucleus bind to KPNB1 and this binding was inhibited by compound 1 which is new candidate drug combination method for cancer therapy. Citation Format: Chan Hee Park, Seung Wook Ham, HyeKyoung Shin, Kyung Soo Oh. Inhibition of kPNB1 and NUPR1 binding increase the anti-cancer drug sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5138.

Small molecule electro-optical binding assay using nanopores

The identification of short nucleic acids and proteins at the single molecule level is a major driving force for the development of novel detection strategies. Nanopore sensing has been gaining in prominence due to its label-free operation and single molecule sensitivity. However, it remains challenging to detect small molecules selectively. Here we propose to combine the electrical sensing modality of a nanopore with fluorescence-based detection. Selectivity is achieved by grafting either molecular beacons, complementary DNA, or proteins to a DNA molecular carrier. We show that the fraction of synchronised events between the electrical and optical channels, can be used to perform single molecule binding assays without the need to directly label the analyte. Such a strategy can be used to detect targets in complex biological fluids such as human serum and urine. Future optimisation of this technology may enable novel assays for quantitative protein detection as well as gene mutation analysis with applications in next-generation clinical sample analysis.

An automated Bayesian pipeline for rapid analysis of single-molecule binding data

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein’s binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

Abstract B12: GABP selectively binds and activates the mutant TERT promoter across multiple cancer types

Abstract Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBM), harbor highly recurrent mutations in the TERT promoter specific to two nucleotide positions. The common mutation sites, G228A and G250A, may create de-novo ETS family transcription factor binding sites, but the precise mechanism of how these mutations confer increased TERT expression has been elusive. Here, we demonstrate the de-novo ETS motif to be critical for mutant TERT activation by site directed mutagenesis. A focused siRNA screen of the many ETS transcription factors expressed in GBM identifies GABPA as the single ETS factor to selectively regulate the mutant but not the wild type TERT promoter. Single molecule binding assays and ChIP-qPCR analysis reveal that GABPA is exclusively recruited to the mutant allele in vitro and in vivo respectively. Furthermore, this allelic recruitment is consistent across four tested cancer types, highlighting a shared mechanism underlying mutant TERT promoter activation. Tandem flanking native ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression and may provide a novel therapeutic target for multiple cancers. Citation Format: Robert J.A Bell, H. Tomas Rube, Alex Kreig, Andrew Mancini, Shaun F. Fouse, Raman P. Nagarajan, Serah Choi, Chibo Hong, Daniel He, Melike Pekmezci, John K. Wiencke, Margaret R. Wrensch, Susan M. Chang, Kyle M. Walsh, Sua Myong, Jun S. Song, Joseph F. Costello. GABP selectively binds and activates the mutant TERT promoter across multiple cancer types. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B12.

GENO-07A MECHANISM OF MUTANT TERT PROMOTER ACTIVATION SHARED ACROSS CANCER TYPES

The human telomerase is an enzyme critical for maintaining telomere length and chromosomal stability in stem cells. The transcriptional regulation of the telomerase reverse transcriptase (TERT) gene, encoding the catalytic subunit of telomerase, is a rate-limiting step in modulating telomerase activity. Although normally silenced in somatic cells, TERT is aberrantly expressed in 90% of aggressive cancers, highlighting this event as a hallmark of tumorigenesis. Reactivating telomerase helps cells with finite lifespan to achieve limitless proliferative potential and bypass cellular senescence induced by DNA replication-associated telomere shortening. Recently discovered non-coding mutations in the TERT promoter are among the most common genetic alterations observed across multiple cancer types. Specifically, one of two positions, G228A or G250A, is mutated in 20% of medulloblastomas, 44% of hepatocellular carcinomas (HCC), 66% of urothelial carcinomas of the bladder, 71% of melanomas, 78% of oligodendrogliomas, and 83% of primary glioblastomas (GBM). The common mutation sites may create de-novo ETS family transcription factor binding sites, but the precise mechanism of how these mutations confer increased TERT expression has remained elusive. Here, a focused siRNA screen of the many ETS transcription factors expressed in GBM identifies GABPA as the single ETS factor to selectively regulate the mutant but not the wild type TERT promoter. Single molecule binding assays and ChIP-qPCR analysis reveal that GABPA is exclusively recruited to the mutant allele in vitro and in vivo respectively. Furthermore, this allelic recruitment is consistent across four tested cancer types, highlighting a shared mechanism underlying mutant TERT promoter activation. Tandem flanking native ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression across multiple cancers.

Sucrose increases the activation energy barrier for actin–myosin strong binding

To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin–myosin (A–M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A–M dissociation, kT (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A–M strong binding by 37±3%, indicating that sucrose inhibits the rate of A–M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A–M rigor binding (71%) are comparable in magnitude to sucrose’s effects on both V (79±33% decrease) and maximal actin-activated ATPase, kcat, (81±16% decrease), indicating that the rate of A–M strong bond formation significantly influences both kcat and V.

A Force Spectroscopy-Based Protein-Ligand Interaction Assay

Binding of small molecules are crucial to the function and folding of many protein machineries inside cell. Thus it is of fundamental importance to measure the binding affinity of small molecule ligands to proteins and reveal the binding mechanism. Here we report a force spectroscopy based single-molecule binding assay that is capable of determining the binding affinity as well as the binding mechanism of ligands to proteins at the single-molecule level. This assay is based on the difference in the mechanical stability of the given protein upon ligand binding. As a proof-of-principle, we use the binding of metal ions, Ni2+, to an engineered metal binding protein, G6-53, as a model system to establish this method. The apo-G6-53 and Ni2+-bound G6-53 exhibit distinct mechanical stability: apo-G6-53 unfolds at around 120 pN while Ni2+-bound G6-53 unfolds at ∼250 pN. Using their characteristic unfolding forces as a reporter, we were able to directly quantify the partitioning of G6-53 between the apo and Ni2+ bound states at different Ni2+ concentration and measure the binding affinity of Ni2+ to G6-53. The distinct unfolding forces of apo and holo forms of G6-53 also allow us to discriminate different species in the process of folding and Ni2+ binding and measure their kinetic evolution. We unfolded G6-53 by force and waited to allow it to fold and bind with Ni2+. We found that the unfolded G6-53 folds to apo form before incorporating Ni2+. The folding rate of G6-53 is independent of Ni2+ concentration, while the binding rate of Ni2+ to apo form of G6-53 is directly proportional to the Ni2+ concentration. Our kinetic data can be fully described using a “folding before binding” model. We anticipate that this novel assay will find unique applications in the study of various protein-ligand interactions.

A functional single-molecule binding assay via force spectroscopy

Protein-ligand interactions, including protein-protein interactions, are ubiquitously essential in biological processes and also have important applications in biotechnology. A wide range of methodologies have been developed for quantitative analysis of protein-ligand interactions. However, most of them do not report direct functional/structural consequence of ligand binding. Instead they only detect the change of physical properties, such as fluorescence and refractive index, because of the colocalization of protein and ligand, and are susceptible to false positives. Thus, important information about the functional state of protein-ligand complexes cannot be obtained directly. Here we report a functional single-molecule binding assay that uses force spectroscopy to directly probe the functional consequence of ligand binding and report the functional state of protein-ligand complexes. As a proof of principle, we used protein G and the Fc fragment of IgG as a model system in this study. Binding of Fc to protein G does not induce major structural changes in protein G but results in significant enhancement of its mechanical stability. Using mechanical stability of protein G as an intrinsic functional reporter, we directly distinguished and quantified Fc-bound and Fc-free forms of protein G on a single-molecule basis and accurately determined their dissociation constant. This single-molecule functional binding assay is label-free, nearly background-free, and can detect functional heterogeneity, if any, among protein-ligand interactions. This methodology opens up avenues for studying protein-ligand interactions in a functional context, and we anticipate that it will find broad application in diverse protein-ligand systems.

Analytical Currents: STED microscopy illuminates vesicle fate | Imaging of the oxidant peroxynitrite in living cells | Solid immersion lenses made from PDMS | Molecular sorting in microfluidic devices | DNA sequencing on a chip | Single-molecule binding assay in femtoliter arrays | Simple derivatization of glycopeptides for MALDI | Direct electrical detection of DNA synthesis

Analytical Currents: STED microscopy illuminates vesicle fate | Imaging of the oxidant peroxynitrite in living cells | Solid immersion lenses made from PDMS | Molecular sorting in microfluidic devices | DNA sequencing on a chip | Single-molecule binding assay in femtoliter arrays | Simple derivatization of glycopeptides for MALDI | Direct electrical detection of DNA synthesis

Single-molecule assays of calmodulin target binding detected with a calmodulin energy-transfer construct.

We have detected single-molecule binding interactions of a target peptide with the calcium-signaling protein calmodulin (CaM) immobilized in an agarose gel, and we have demonstrated the application of a single-molecule binding assay to measure the binding strength of CaM with the CaM-binding domain of calmodulin-dependent protein kinase II (CaMKII). The results demonstrate the potential for ultrasensitive assays of CaM-target interactions and the measurement of a picomolar dissociation constant. To detect single-molecule protein interactions, single-molecule assays require that the analyte molecule be confined to the focal spot of the objective for the time scale of the measurement. We demonstrate the deleterious effect of surface immobilization on CaM. As an alternative to surface immobilization, we have constructed a CaM/maltose binding protein fusion protein, which renders CaM translationally immobile in a low weight percent agarose gel. The target binding functionality of CaM assayed in agarose gels is in good agreement with solution assays. The utility of the construct for detecting interactions with CaM targets was demonstrated in a single-molecule assay of binding interactions of MBP-CaM with the CaMKII CaM-binding domain peptide. A value of 103 +/- 35 pM for the dissociation constant of this interaction was determined by simple counting of fluorescent molecules.