This research was supported by a grant from National Science Foundation (MCB 1818310) to RAF.&#160;This work was supported in part by the Chemical Biology Core Facility/Protein Crystallography Unit at the H. Lee Moffitt Cancer Center (NIH/NCI: P30-CA076292).

1. Preparation of materials
<ol>
	<li>Prepare phosphate buffered saline (PBS): 137 mM NaCl, 2.5 mM KCl, 10 mM NaH2PO4, and 2 mM KH2PO4, pH 7.41.</li>
	<li>Prepare NTA-Atto 647 N dye. Dilute stock NTA-Atto 647 N dye to 100 nM from a 100% DMSO solution into PBS without Tween.</li>
	<li>Express Vam7-His8 &#8211; Protein as a fusion protein in E. coli and purify using Ni-NTA and size exclusion chromatography8.</li>
	<li>Tit...

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	<li>Jerabek-Willemsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroScale+Thermophoresis:+Interaction+analysis+and+beyond.">MicroScale Thermophoresis: Interaction analysis and beyond.</a> Journal of Molecular Structure. 1077, 101-113 (2014).</li><li>Chapman, D., Hochstrasser, R., Millar, D., Youderia, n. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+proteins+without+primary+sequence+tryptophan+residues:+mutant+trp+repressors+with+aliphatic+substitutions+for+tryptophan+side+chains.">Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.</a> Gene. 163 (1), 1-11 (1995).</li><li>Duff, M. R., Grubbs, J., Howell, E. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+microscale+thermophoresis+discriminates+sites+and+affinity+of+protein-ligand+binding.">Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.</a> Angewandte Chemie International Edition English. 51 (42), 10656-10659 (2012).</li><li>Starr, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidic+acid+induces+conformational+changes+in+Sec18+protomers+that+prevent+SNARE+priming.">Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.</a> Journal of Biological Chemistry. 294 (9), 3100-3116 (2019).</li><li>Miner, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Central+Polybasic+Region+of+the+Soluble+SNARE+(Soluble+N-Ethylmaleimide-sensitive+Factor+Attachment+Protein+Receptor)+Vam7+Affects+Binding+to+Phosphatidylinositol+3-Phosphate+by+the+PX+(Phox+Homology)+Domain.">The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.</a> Journal of Biological Chemistry. 291 (34), 17651-17663 (2016).</li><li>Collins, M. D., Gordon, S. 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The determination of Vam7-His8binding to DiC8-PA provided a robust fitted KD&#160;for the given interaction, which is slightly lower affinity than the measured KD of Vam7-His8&#160;to PA liposomes (unpublished). This difference is most likely due to the lack of a membrane, which generally results in lower affinity for membrane specific lipid binding interactions and therefore demonstrates the role for the liposome membrane scaffold to this interaction<sup class="x...

Microscale thermophoresis is a relatively new technique in determining disassociation constants (KD) as well as inhibition constants (IC50) between biochemically relevant ligands. The leading commercial retailer for MST (e.g., NanoTemper) offers two popular MST technologies: 1) Label free MST requiring a fluorescent tag, and 2) labeled thermophoresis using the inherent fluorescence of proteins dependent on the number of aromatic residues present in each protein1. A disadvantage of label-free thermophoresis is that in most cases, it does not allow for the measurement of protein-protein interactions. However, it may be possible to engineer proteins without aromatic amino acids such as tryptophan for use in label free thermophoresis2.
MST measures the movement of particles in response to the induction of microscopic temperature fields initiated by an infrared laser in currently available technologies1. MST can be used to measure protein-protein interactions, protein-lipid interactions, protein-small molecule, competition experiments, and even interactions between small-molecules so long as one can produce enough signal separation. Additionally, MST allows for the measurement of membrane-protein based interactions embedded in either liposomes or nanodiscs. Labeled thermophoresis takes advantage of the use of fluorescently labeled tags allowing for chemically controllable separation of signal between ligand and analyte. KD values can be obtained using thermophoresis for interactions involving protein binding at low nanomolar concentrations, which in most cases is a much lower concentration of protein than what is required for isothermal calorimetry (ITC)3. Additionally, MST does not have strict buffering requirements as required for surface plasmon resonance (SPR)4 and labeled thermophoresis can even be used to measure binding constants of proteins of interest from non-fully purified protein solutions5 with genetically inserted fluorescent tags6. A disadvantage of MST is that kinetic parameters cannot be obtained readily for MST as in SPR2.
Thermophoresis measurements depend on the local temperature difference of a solution. This heat can be generated from an infrared laser. The MST device has a fluorescence detector coupled to an infrared (IR) beam and can pick up changes in fluorescence from local concentration changes of the fluorescent molecules at the point where the IR laser is targeted. The MST device utilizes an IR targeted laser coupled directly to a fluorescence detector focused at the same point in which the heat is generated in the solution. This allows for robust detection of changes in temperature corresponding to the depletion of molecules at the point of heat generated by the IR laser. Measured fluorescence generally decreases closer to the IR laser in response to temperature increases. The differences measured as a result can be due to multiple factors including charge, size, or solvation entropy. These differences are measured as changes in fluorescence in response to induction of heat or movement of molecules from hot to colder parts of the capillary.
When loading a capillary with a given solution, it is important to leave air at either end of the capillary and not load the capillary completely full. The commercial capillary holds about 10 &#956;L of solution. One can achieve accurate measurements with 5 &#956;L of solution so long as the solution is manipulated to the center of the capillary, there are no air bubbles (potentially degas prior to loading capillary), and one is careful loading the rack to not jostle the solution from the center of the capillary, where the infrared laser is targeted. If the laser does not come in contact fully with solution, the result will most likely be one of three unusable outputs for that concentration: 1) no or low fluorescence detection, 2) higher fluorescence detection (potentially with jagged peak), or 3) fluorescence detection within other values from given titration, but with a jagged and unrounded peak.
For labeled thermophoresis it is optimal to have a fluorescence signal above 200 and below 2000 fluorescence units7. The MST device uses a range of LED intensities from 0 to 100, which can be selected to achieve a signal above 200 or below 2000. Alternatively, one can use different concentrations of the labeled ligand to modify the fluorescence signal to an optimal level. It is important to run a cap scan with a given MST measurement as a reference when analyzing data, as a poor cap scan can often result in a point that may later be determined to be an outlier. Each run should take approximately 30 min if measuring a single MST power with a cap scan. The commercial devices allow changes in MST power. In older software versions this could be set from 0 to 100; and in later versions one can select low, medium, or high MST. To achieve robust traces, a researcher may need to try each of these and decide which MST setting results in the most robust data for a given interaction.

<table>
	<tbody>
		<tr>
			<td>Cy5 Maleimide Mono-Reactive Dye</td>
			<td>GE Healthcare</td>
			<td>PA23031</td>
			<td>For protein labeleing</td>
		</tr>
		<tr>
			<td>Graphpad Prsim</td>
			<td>Graphpad software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monolith NT.115 Capillaries (1000 count)</td>
			<td>Nanotemper</td>
			<td>MO-K022</td>
			<td>Capillaries for MST</td>
		</tr>
		<tr>
			<td>Monolith NT.115 machine</td>
			<td>Nanotemper</td>
			<td></td>
			<td>University equipment</td>
		</tr>
		<tr>
			<td>NTA-Atto 647 N</td>
			<td>Sigma</td>
			<td>2175</td>
			<td>label for His tags</td>
		</tr>
		<tr>
			<td>Phosphatidylinositol 3-phosphate diC8 (PI(3)P diC8)</td>
			<td>Echelon</td>
			<td>P-3008</td>
			<td>Lipid for binding experiments</td>
		</tr>
	</tbody>
</table>

use of microscale thermophoresis to measure protein-lipid interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

This is a sample output using the affinity analysis. The labeled MST was used to determine the binding constant of the Vam7-His8&#160;to the soluble dioctanoyl (DiC8) PA of one of its natural substrates9. Figure 1 presents the thermophoretic traces from one trial of a 1:1 titration of DiC8 PA starting at 500 &#956;M against 50 nM of Vam7-His8. Initial fluorescence (time before infrared laser turned on), Tjump (time initially after infrared laser turned on), and thermo...

Watch this Scientific Journal Video about Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions at JoVE.com

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Labeled microscale thermophoresis obtains disassociation constants, or KDs, of diverse biochemical interactions efficiently. This protocol and the accompanied video give the approximate affinity for Vam7, the only soluble SNARE protein in yeast, to phosphatidic acid. This is the way that Vam7 associates with two membranes before interacting with other proteins.This method obtains KDs rapidly for diverse biochemical interactions at low cost, with high reproducibility and with minimal protein cost. This technique is suitable for first-stage pharmaceutical development, allowing for readily obtainable KDs to determine potential lead compounds. Before beginning an analysis, turn on the power switch on the back of the MST device and open the control software.Confirm that the computer is attached to the device and in connected status and set the MST Before to 3 seconds, the MST to 30 seconds, and the Fluorescence Recovery to after 1 second. For each capillary tube, enter the name of the target ligand, the name of the ligand analyte, the concentration of the target, and the highest titration concentration using the autofill titration ratio. Select a range of MST powers and enter values for each power to test for the most robust binding fit.To prepare labeled MST samples, dilute a Vam7-octahistidine solution to a 200 nanomolar concentration and an NTA-Atto 647 dye to 100 nanomolar concentration, both in PBS. Mix the Vam7-octahistidine and the NTA-Atto 647 dye at a 1:1 volumetric ratio and allow the mixture to sit at room temperature protected from light for 30 minutes. At the end of the incubation, centrifuge the dye and protein mixture and store the solution at 4 degrees Celsius for up to a few hours before use.Then, prepare the sample, take dyed ligand and expose the analyte. For microscale thermophoresis of the sample, open the device and slide the capillary rack out. Load the sample capillaries into the rack with the highest concentration at position one and select the Red channel in the control software.Then, load the rack in the instrument. Then select a range of MST power and start the Cap Scan MST Measurement and evaluate the shift response. Thermophoretic traces from one trial of a 1:1 titration of DiC8 PA starting at 500 micromoles against 50 nanomolar NTA-Atto 647 labeled Vam7 domain are shown here.The initial fluorescence, time jump, and thermophoresis can be observed, allowing KD to be calculated from any one or a combination of these measurements. A saturation curve can be plotted from these results, for example, for the thermophoresis with the time jump output, as illustrated in the graph. Generally, MST results are determined using log scale, taking into account the protein concentration to determine the binding affinity by selecting the KD model and inputting the protein concentration.When performing this protocol, ensure that the solution is in the center of the capillary and that there's no air bubbles and that the capillary does not have dust or solution on the outside of the capillary. Isothermal titration calorimetry or surface plasmon resonance could be used to validate the experimental KD obtained. If there is potential cooperative binding, ITC would be the next logical method performed, given resources.This technique has enabled traditional biology researchers to incorporate biophysical screening techniques into their research for determining pathway relationships. One example of this technique is cell lysase with the target protein expressed endogenously, where the GFP tag is a novel way to perform a traditional pull-down assay.

use-of-microscale-thermophoresis-to-measure-protein-lipid-interactions

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JoVE Journal

Biochemistry

6.3K visualizações.  University of Illinois at Urbana-Champaign. A termoforese de microescala rotulada obtém constantes de dissociação, ou KDs, de diversas interações bioquímicas de forma eficiente. Este protocolo e o vídeo acompanhado dão a afinidade aproximada por Vam7, a única proteína snare solúvel na levedura, ao ácido fosfídico. Esta é a maneira que Vam7 associa com duas membranas antes de interagir com outras proteínas.Este método obtém KDs rapidamente para diversas interações bioquímicas a baixo custo, com alta reprodutib...