Name: bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects
Uploaded: 2014-02-18T14:15:00
Description: Watch this Scientific Journal Video about Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects at JoVE.com

We thank Fort&#233;Bio for providing graphics used in Figure 1. This work was supported by NIH grant GM083088 to T.M.D.

1. Programming the Instrument for BLI Assay
Turn on the instrument at least one hour in advance to allow the lamp to warm up; this is necessary to minimize noise and drift in optical signal during the experiment. Set the desired temperature via the instrument tab to prewarm the sample plate holder. Then set up the experimental design in the Data Acquisition software. Select "New Kinetics Experiment" in the Experiment Wizard tab. This presents a tabbed menu with all steps that must be defined.</p...

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Currently available instruments for BLI allow significant throughput and flexibility in assays for biomolecular interactions. Various solution samples are dispensed in wells of a black microtiter plate, and a set of parallel BLI sensors are programmed to move back and forth between columns of wells on the plate. The samples are stirred by orbital shaking throughout the assay. The system used here has 8 sensors and uses a 96-well sample plate, but another system uses 16 sensors and a 384-well sample plate. Thus, interacti...

Protein-protein interactions are important for many biological processes, and label-free optical methods like Surface Plasmon Resonance (SPR) have been used in vitro to study kinetics of binding and dissociation1. Most label-free methods immobilize one biomolecule on a sensor surface and use an optical signal to detect a binding partner from solution as it associates with the immobilized biomolecule1. While SPR is a highly sensitive method, it is prone to interference due to changes in the refractive index of the solution flowing over the sensor2. Although not as sensitive as SPR, Bio-layer Interferometry (BLI) is less affected by changes in sample composition1,3. BLI uses fiber optic biosensors that have a proprietary biocompatible coating at the tip. The system used here (Octet-RED96) contains eight spectrophotometers. White light is piped to a row of probes that move on a robotic arm. Fiber optic sensors are picked up by the probes and moved to a 96-well plate containing samples. One of the target molecules is immobilized on the biosensor surface. Then sensors are moved to wells containing the binding partner in solution. BLI monitors association of the binding partner with the immobilized molecule, and then monitors dissociation after moving the sensors to solution without the binding partner. Binding of molecules to the biosensor surface leads to changes in optical interference between light waves that reflect back to the spectrophotometers from an internal surface and from the external interface between sensor and solution. These changes in interference can be quantified and used to determine kinetic rates of binding and dissociation, as summarized in the animation of Figure 1.
We have applied BLI to measure interactions between the catalytic complex of bacterial ATP synthase and its &#949; subunit, which can auto-inhibit the enzyme. ATP synthase is a membrane-embedded rotary nanomotor that catalyzes synthesis and hydrolysis of ATP4.&#160;The catalytic complex (F1) can be isolated in a soluble form that works as an ATPase. Subunit &#949; has two domains: the N-terminal domain (NTD) is necessary for proper assembly and functional coupling of the enzyme but does not interact directly with the catalytic subunits; the C-terminal domain (CTD) can inhibit the enzyme by interacting with multiple catalytic subunits5,6. This &#949;-mediated regulation is specific to bacterial ATP synthases and is not observed in the mitochondrial homologue. ATP synthase has emerged as a target for antibacterial drugs, as shown by recent FDA approval of bedaquiline to treat drug-resistant tuberculosis7. Thus, targeting &#949;&#8217;s inhibitory role for drug discovery could yield antibacterials that do not inhibit the mitochondrial ATP synthase. With the isolated catalytic complex (F1), &#949; becomes a dissociable subunit. However, with &#949; bound to F1, the &#949;CTD can undergo a dramatic conformational change, partially inserting into the enzyme&#8217;s central cavity and forming an inhibitory state that is unlikely to dissociate directly6,8. We use BLI to measure kinetics of F1/&#949; binding and dissociation, and indirectly, to examine allosteric effects of catalytic-site ligands on &#949;&#8217;s conformation.
In our system, &#949; was chosen for immobilization on the sensor surface since BLI signal (like SPR) is sensitive to the mass of the molecules binding at the surface. The &#949; subunit is small (~15 kDa) relative to the main F1 complex (~347 kDa). Thus, a larger BLI signal will result from binding of F1 to immobilized &#949;. In order to monitor F1 dissociation, which can be very slow,&#160;&#949;&#160;must be strongly immobilized. Thus we chose to biotinylate&#160;and immobilize it on streptavidin-coated biosensors.&#160;Proteins can be biotinylated by (i) random modification of surface lysines9, (ii) reaction of a unique native or engineered cysteine with a biotin-maleimide reagent10 or (iii) genetically adding a specific biotin-acceptor peptide that is enzymatically biotinylated during in vivo expression of the tagged protein11. In our system, &#949; is biotinylated using method (iii)8. Once biotin-tagged &#949; is immobilized on streptavidin sensors, BLI can measure the binding and dissociation of F1 that has been depleted of subunit &#949; (F1(-&#949;)). For the experiments described here, preliminary assays had been done to determine reasonable amounts of the biotinylated protein to immobilize on the sensors. This can vary, depending on the molecular weight of the protein and its binding partner, but the goal is to determine a minimal amount of immobilized protein that provides (i) acceptable signal-to-noise for binding kinetics with a low concentration of the binding partner (below KD) and (ii) minimal distortion of binding kinetics with near-saturating concentration of the binding partner. Also, stoichiometry of biotinylation may vary (but avoid &#62;1 mol biotin/mol protein), so some initial assay may be needed for each new lot of biotinylated protein to confirm that a consistent BLI signal can be achieved during immobilization on the streptavidin-coated sensors.

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			<td height="21" style="height:21px;width:216px;">Octet-RED96</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">30-5048</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">A6003-10G</td>
			<td>Fatty Acid free</td>
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			<td height="21" style="height:21px;width:216px;">Biosensor/Streptavidin</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">18-5019</td>
			<td>Tray of 96 sensors</td>
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		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Microtiter plate</td>
			<td style="width:108px;">Greiner Bio-one</td>
			<td style="width:119px;">655209</td>
			<td>Black, Polypropylene</td>
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			<td height="21" style="height:21px;width:216px;">Data Acquisition software</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">Version 6.4</td>
			<td>Newer versions available</td>
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			<td height="21" style="height:21px;width:216px;">Data Analysis software</td>
			<td style="width:108px;">Pall/Fort&#233;Bio</td>
			<td style="width:119px;">Version 6.4</td>
			<td>Newer versions available</td>
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bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

Real time binding and dissociation BLI kinetics are shown in Figure 3. This experiment was done with assay buffer in association and dissociation. This experiment was started with a 10 min baseline step since the sensors had been prewet only briefly. Next, biotinylated &#949; was loaded on the sensors. No detectable dissociation of &#949; occurred throughout all remaining steps as seen from the reference curve (G) which had no binding partner added. A second reference sensor (H) was devoid of immobilized...

Watch this Scientific Journal Video about Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects at JoVE.com

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

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Research

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Chemistry

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature solvents. These coordinating ligands help solubilize and stabilize the nanoparticles while in solution, and can influence the resulting size and reactivity of the nanoparticles during their formation. Despite the ubiquity of using ligands during synthesis, the presence of aliphatic ligands on the nanoparticle surface can result in a number of problems during the end use of the nanoparticles, necessitating further ligand stripping or ligand exchange procedures. We have developed a way to synthesize cadmium sulfide (CdS) nanoparticles using a unique sulfur copolymer. This sulfur copolymer is primarily composed of elemental sulfur, which is a cheap and abundant material. The sulfur copolymer has the advantages of operating both as a high temperature solvent and as a sulfur source, which can react with a cadmium precursor during nanoparticle synthesis, resulting in the generation of ligand free CdS. During the reaction, only some of the copolymer is consumed to produce CdS, while the rest remains in the polymeric state, thereby producing a nanocomposite material. Once the reaction is finished, the copolymer stabilizes the nanoparticles within a solid polymeric matrix. The copolymer can then be removed before the nanoparticles are used, which produces nanoparticles that do not have organic coordinating ligands. This nascent synthesis technique presents a method to produce metal-sulfide nanoparticles for a wide variety of applications where the presence of organic ligands is not desired.

Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction.

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and &#946; liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Accessing Valuable Ligand Supports for Transition Metals: A Modified, Intermediate Scale Preparation of 1,2,3,4,5-Pentamethylcyclopentadiene

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that derive from initial 2-bromo-2-butene lithiation followed by acid mediated dienol cyclization. The revised synthesis and purification of the ligand avoids the use of mechanical stirring while still permitting access to significant quantities (39 g) of Cp*H in good yield (58%). The procedure offers other additional benefits, including a more controlled quench of excess lithium during the production of the intermediate heptadienols and a simplified isolation of Cp*H of sufficient purity for metallation with transition metals. The ligand was subsequently used to synthesize [Cp*MCl2]2 complexes of both iridium and ruthenium to demonstrate the utility of the Cp*H prepared and purified by our method. The procedure outlined herein affords substantial quantities of a ubiquitous ancillary ligand support used in organometallic chemistry while minimizing the need for specialized laboratory equipment, thus providing a simpler and more accessible entry point into the chemistry of 1,2,3,4,5-pentamethylcyclopentadiene.

Reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented. The revised protocol for the synthesis and purification of the ligand minimizes the need for specialized laboratory equipment while simplifying reaction workups and product purification. Use of Cp*H in the synthesis of [Cp*MCl2]2 complexes (M = Ru, Ir) is also described.

A reliable, intermediate scale preparation of 1,2,3,4,5-pentamethylcyclopentadiene (Cp*H) is presented, based on modifications of existing protocols that ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.