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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The authors declare that they have no conflicts of interest.

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.

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
	</tbody>
</table>

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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Chemistry

28.6K visualizações.  SUNY Upstate Medical University.  Os protocolos aqui descrever os ensaios cinéticos de interações proteína-proteína com Bio-camada Interferometria. Tipo F ATP sintase, que está envolvida no metabolismo da energia celular, pode ser inibida pela sua subunidade ε em bactérias. Temos adaptado Bio-camada de interferometria para estudar as interacções do complexo catalítico com o domínio C-terminal do inibidor ε. 