This project has been supported by NSERC RGPIN-2018-04994, Campus Alberta Innovation Program (RCP-12-002C) and Alberta Prion Research Institute / Alberta Innovates Bio Solutions (201600018), awarded to M.O and Genome Canada and Canada Foundation for Innovation grants awarded to The Metabolomics Innovation Centre (TMIC) and NANUC.

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The authors disclose no conflicts of interest.

The 2D 15N-resolved NMR experiment is one of the most widely used methods to show how two molecules interact. It is the most information-rich method that allows both partners&#39; signals to be continuously monitored throughout a titration experiment in solution state. Although typically qualitative in the case of large complexes, the method can also be used in favorable cases to measure binding affinities where NMR signals can be tracked in high resolution spectra. Where assignments can be conveniently made, such as in the case of many proteins under 20 kDa in size, the binding sites can also be mapped. Complementary assays such as MST provide quantitative information about interactions in solution, and require less protein in unlabeled states. Comparison of mutant binding data is useful for providing controls to ensure that interactions evidenced by NMR line broadening are genuine and not artifacts of, for example, aggregation or viscosity changes.
Protein Expression
Streamlining the expression process reduces the amount of labor intensive protein production. Part of this optimization process involves identification of an appropriate strain of E. coli for the recombinant expression of protein. Strain preference depends on elements including the nature of the vector in use and, more specifically, the ultimate stability of the recombinant protein being expressed7. The risk of degradation of the heterologous protein by endogenous E. coli proteases can be reduced by use of protease deficient E. coli such as the BL21 strain. For genes containing rare codons, a strain such as BL21-CodonPlus (DE3)RIPL may be preferred. This strain combines the protease deficient nature of the BL21 strain with additional endogenous copies of rare codon tRNAs for arginine, isoleucine, proline, and leucine. Alternatively, rare codons that can compromise overexpression may be avoided by ordering a codon-optimized construct from a commercial source. Many strains of E. coli are available for recombinant gene expression, each optimized for circumvention of a particular problem during expression7. In the case of this study, the standard protease deficient strain BL21(DE3) produced adequate quantities of soluble protein for subsequent purification and analysis.
Protein Purification
The purification protocol for a given protein is often unique in the sense that each protein remains stable and soluble under different conditions such as temperature, salt concentration, or pH. The overall effectiveness of purification through affinity chromatography is also sensitive to the concentration of eluting species such as imidazole at various steps during the purification process. In this work, critical buffer conditions for IMAC were pH for the E-PRD, and imidazole concentration for the VimRod. A pH of 7.5 was required to avoid precipitation of the E-PRD following initial elution from the IMAC column. For the IMAC purification of VimRod, increasing the concentration of imidazole from 30 to 50 mM during the column wash step was found to have a substantial improvement in the purity of the final elution fractions. For the elution step, increasing the concentration of imidazole from 250 to 350 mM also was found to improve the yield of the final elution. Initial attempts to elute protein using 250 mM imidazole led to incomplete elution of VimRod as revealed by a final 1 M imidazole strip of the column (data not shown). Increasing the imidazole concentration to 350 mM for the elution was sufficient to recover all of the protein bound to the column. S can serve a dual purpose because it acts as a polishing step for protein purification while simultaneously performing buffer exchange. Buffer exchange is a critical step for subsequent binding analysis since it removes the imidazole used to elute His6-tagged protein. It also serves as an opportunity to change conditions such as salt concentration or pH, which may impact the efficacy of certain downstream techniques or assays. Protein thermal shift (PTS) can be used to identify optimal buffers for downstream assays, especially for those requiring stable protein for prolonged periods of time at room temperature8,9.
Binding Analysis
Protein that is freshly prepared is critical for accurate binding assays, although frozen protein can also be used as long as the results are compared. Filamentous proteins such as vimentin multimerize in a salt and pH dependent fashion, and hence the solution condition need to be optimized and the oligomeric state estimated by a method such as SEC10,11, dynamic light scattering12 or analytical ultracentrifugation13,14,15. NMR spectroscopy is well suited for measuring ligand interactions of small proteins at atomic resolution. However, when a protein interacts with larger molecule, slower tumbling ensues, and this results in loss of signals, which can confirm binding although it does not necessarily allow mapping of binding sites, which would also require assignment of at least backbone resonances. In this scenario, NMR experiments do not allow identification of the interaction site. Hence site directed mutagenesis is applied to identify the critical residues needed for the binding. Such mutants therefore do not exhibit signal loss. In this protocol, a mutant form with a substitution at position 1914 retains the peak intensities in presence of VimRod and therefore confirms disruption of the interaction of E-PRD and VimRod. Assignment of the backbone and sidechain resonances would add value to this approach, particularly as the structure for the free E-PRD has been solved by X-ray crystallography4. Future applications of NMR include characterization of complex interactions between larger molecules and will benefit from ultra high field magnets and the use of other observable groups such as 13C-labeled and trifluoro methyl groups as reporters.
MST has a number of advantages for studying binding interactions16. The binding partners are free in solution and not immobilized. Analysis of the quality of the samples is built into the software with quality control reporting of aggregation, adsorption to the capillaries or insufficient fluorescent labeling of the target molecule. Small amounts of the target are typically used, the concentration of the labeled target is usually between 20-50 nM in a 10-20 &#956;L volume/reaction. This protocol uses very small reaction volumes (10 &#956;L) to maximize the concentration of ligand that can be achieved in the titrations allowing weak binding interactions to be characterized. This necessitates accurate pipetting and care being taken to avoid introducing bubbles while still thoroughly mixing. Adequate mixing is critical for accurate, consistent fluorescence measurements along the set of serial dilutions. The amount of Tween-20 in the MST experiments was reduced from a standard 0.05% to 0.015% to lower the tendency to create bubbles and improve mixing.
The RED-Tris-NTA dye provides a quick, easy and convenient way to fluorescently label any protein that has a His tag. The labeling is effectively complete in only 30 minutes and is very tight so that no dye removal procedure is necessary. No modifications are made to amino acid residues in the protein that might alter the ligand binding properties. A caveat is that only the protein to be labeled should have a His6 tag. This required the cleavage of the tag from the ligand protein, E-PRD, and the removal of the tag and uncleaved E-PRD with a second IMAC column step. If possible, the ligand protein should be prepared without the use of a His tag. Alternatively, proteins may be covalently labeled with a fluorophore through amine coupling to lysine residues or thiol coupling to cysteine residues. However, care must be taken when using such systems since the covalent attachment of a fluorophore may affect electrostatic or polar binding interactions relying on lysine or cysteine residues. The quantification of binding affinity between the VimRod and E-PRD by MST was unusually sensitive to salt concentration. This problem was mitigated by initially dialyzing both the target and ligand into the same batch of assay buffer. Nonetheless, saturation of the MST binding curve could not be achieved when performing the MST assay in the presence of 150 mM NaCl due to the complex behavior of VimRod. Reliable, complete data was obtained once the concentration of NaCl was lowered to 10 mM allowing accurate calculation of the KD. Hence, careful optimization of solution conditions and comparison with complementary assays are recommended to achieve robust results. Furthermore, MST may be used to quantify the salt dependence for a given interaction, quantify stoichiometric properties of protein interactions, monitor protein folding, and probe into enzyme kinetics17.

Interactions between proteins allow the formation of molecular machines that create order within cells. The individual interactions are often weak but usually contribute to multivalent complexes that can be cooperative and dynamically regulated. Sensitive assays that provide atomic resolution and quantitative information about such complex interactions are needed to deduce mechanisms and design interventions such as drug-like molecules. NMR spectroscopy is an efficient method for obtaining such information about protein interactions, and also is used for fast screening for ligands including those that bind weakly1. The NMR methods used can be categorized into those that are protein observe or ligand observe. This manuscript uses the former approach in which a spectrum of a stable-isotope labeled protein that is comparatively small (usually under 20 kDa) is acquired and the unlabeled ligand is titrated. This allow the labeled residues involved in the interaction to be mapped in favorable cases. Once the complex forms, there are changes in the chemical environments of interacting residues that manifest themselves as changes in the chemical shift and shape of their NMR signals. The extent of such changes correlates with the degree of involvement of these groups in the interaction. Chemical shift perturbations (CSPs) can be measured by comparing a series of NMR spectra of the protein collected in the absence and presence of varying amounts of the ligand. For larger ligands or complex interactions, the change in peak shape or intensity can be measured to deduce interactions.
The most common 2D experiment used for detecting ligand interactions is the 15N-heteronuclear single quantum correlation (HSQC) experiment2. This requires that one protein be uniformly labeled with 15N, which is typically achieved by expressing them as affinity-tagged versions in E. coli bacterial cultures grown in 15N-enriched media. Binding is apparent when the HSQC spectra collected during the titration are superimposed, revealing peak changes for a subset of residues involved in the complex formation. The interaction can occur in the fast exchange regime where the free and ligand-saturated state signals collapse into one population averaged peak. Alternatively, in the case of slow exchange between the states, both signals are observed with integrals that represent their relative amounts. While NMR lineshape analysis can be used to estimate the binding affinities in some cases, methods such as MST have also proven convenient and provide cross-validation of genuine interactions.
The example provided is of two proteins found within desmosomes. They mediate junctions between cell surfaces and the cytoskeleton and mediate multivalent interactions between cell adhesion machines and intermediate filaments to maintain the integrity of skin and heart tissues and withstanding of shear forces. Diseases can result when desmosomal proteins such as desmoplakin or vimentin are compromised by mutations or autoantibodies, leading to destabilization of cell-cell junctions, and hence their interactions are of critical importance3. The structural basis of ligand binding by desmosomal proteins can be characterized by NMR spectroscopy, while the interactions can be quantified by MST. Methods herein were used to characterize the interactions between plakin repeat domains (PRDs) which often are present as tandem sets that offer basic grooves, and vimentin, an intermediate filament that interacts through an acidic surface offered by its helical bundle4. These complexes are formed at the cell membrane where they anchor for intermediate filaments of the cell cytoskeleton to desmosomes that connect to adjacent cells, thus forming a network of adhesive bonds that radiates throughout a tissue.

<table><tbody><tr><td>Monolith NT.115, includes control and analysis software</td><td>NanoTemper Technologies</td><td>MO-G008</td><td>Instrument for microscal thermophoresis</td></tr><tr><td>His-Tag Labeling Kit RED-Tris-NTA</td><td>NanoTemper Technologies</td><td>MO-L008</td><td>RED-tris-NTA dye for MST</td></tr><tr><td>Standard capillaries</td><td>NanoTemper Technologies</td><td>MO-K022</td><td>capillaries for MST</td></tr><tr><td>HisTrap HP, 5 mL</td><td>GE Healthcare</td><td>17524801</td><td>IMAC column</td></tr><tr><td>HisPur Ni-NTA resin, 100 mL</td><td>Thermo Fisher Scientific</td><td>88222</td><td>IMAC resin</td></tr><tr><td>HiLoad 16/600 Superdex 75 pg</td><td>GE Healthcare</td><td>28989333</td><td>SEC column</td></tr><tr><td>TEV protease</td><td>Sigma Aldrich</td><td>T4455</td><td>cleavage of his tag from E-PRD</td></tr><tr><td>BL21(DE3) Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2527H</td><td>cells for protein expression</td></tr><tr><td>cOmplete Protease Inhibitor Cocktail Tablets EDTA-Free</td><td>Sigma Aldrich</td><td>11873580001</td><td>protease inhibitors for protein purification by IMAC</td></tr><tr><td>TCEP, Tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma Aldrich</td><td>C4706</td><td>reducing agent for protein purification</td></tr><tr><td>Reagents (HEPES, NaCl, etc)</td><td>Sigma Aldrich</td><td>various</td><td>Preparation of media and buffers</td></tr><tr><td>Ammonium chloride (&lt;sup&gt;15&lt;/sup&gt;N, 99%)</td><td>Cambridge Isotope Laboratories</td><td>NLM-467</td><td>isotope labelling for NMR</td></tr><tr><td>D&lt;sub&gt;2&lt;/sub&gt;O, Deuterium Oxide (D, 99.8%)</td><td>Cambridge Isotope Laboratories</td><td>DLM-2259</td><td>NMR sample preparation</td></tr><tr><td>DSS, Sodium 2,2-dimethyl-2-silapentane-5-sulfonate-D6 (D,98%)</td><td>Cambridge Isotope Laboratories</td><td>DLM-8206</td><td>reference for NMR</td></tr><tr><td>Precision 5 mm NMR Tubes, 7&amp;rdquo; long</td><td>SJM/Deuterotubes</td><td>BOROECO-5-7</td><td>NMR tubes</td></tr><tr><td>NMR spectrometer (14.1 Tesla)</td><td>Bruker</td><td></td><td>acquisition of NMR data</td></tr><tr><td>TCI 5mm z-PFG cryogenic probe</td><td>Bruker</td><td></td><td>acquisition of NMR data</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bruker TopSpin 4.0.1</td><td>Bruker</td><td></td><td>processing of NMR data</td></tr><tr><td>MO.Control</td><td>NanoTemper Technologies</td><td></td><td>included with Monlith NT.115</td></tr><tr><td>MO.Affinity Analysis</td><td>NanoTemper Technologies</td><td></td><td>included with Monlith NT.115</td></tr></tbody></table>

measuring interactions of globular and filamentous proteins by nuclear magnetic resonance spectroscopy (nmr) and microscale thermophoresis (mst)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

The E-PRD domain (residues 1822-2014 cloned into pProEX-HTC) of the human envoplakin gene and the VimRod domain (residues 99-249 cloned into pET21a) of human vimentin4 were expressed with His6 tags and purified. Figure 6 and Figure 7 demonstrate the levels of purity of VimRod (18.8 kDa) and E-PRD (21.8 kDa) obtained from this method of protein purification. The removal of the His6 tag from the E-PRD construct is essential for the MST experiments as the VimRod protein is labeled using a His6 tag binding dye and any E-PRD retaining its His6 tag may compete for binding of the dye. The second IMAC column after cleavage of the tag with TEV protease removes the TEV protease, the cleaved tag and any uncleaved His6-E-PRD that remained. The final polishing step of the purification is size exclusion chromatography. Despite both proteins being of a similar size, the VimRod elutes from the column at a 51 mL while the E-PRD elution peak is centered at 72 mL where a protein monomer of this size would be expected. The apparent increase in size of VimRod is likely due to the its characteristics as a filamentous long rod shaped protein as analytically ultracentrifuge experiments demonstrated that VimRod was monomeric4. Lower yields of protein are obtained from the cultures grown in M9 than those from rich broth due to a lower amount of cells being produced in the minimal media. The initial growth of larger starter cultures for M9 preparations in TB allows improvement of cell yields while maintaining the extent of 15N labeling necessary for the NMR experiments.
The 15N-1H HSQCs were acquired for the wild type and R1914E mutant of E-PRD in presence or absence of VimRod (Figure 8A-8D). The spectrum of E-PRD in Figure 8A shows the expected number of well resolved peaks, indicative of a properly folded protein. In the presence of VimRod (Figure 8B) the spectrum shows extensive line broadening and peak disappearance, corresponding to binding between the E-PRD and VimRod. This binding is lost by mutation of R1914E as evidenced by comparison of Figure 8C and 8D. Little change is observed in the spectrum upon addition of VimRod to the R1914E mutant indicating a lack of binding between this mutant E-PRD and VimRod. The E-PRD peak intensities in the presence/absence of VimRod were compared and plotted as the relative peak intensities in Figure 8E, which indicates the range of peak broadening in the E-PRD complex. The R1914E mutant of E-PRD (not shown) retained about 97% of peaks at 20% or higher peak intensities in presence of VimRod compared to about 20% for the wild type (Figure 8E). This represents a loss of function point mutant, with additional mutants having intermediate effects also having been studied4.
To validate and quantitate the binding of VimRod and E-PRD MST analysis using His6-VimRod labeled with fluorescent RED-tris-NTA dye as the target mixed with decreasing concentrations of the ligand E-PRD from 1.28 mM to 39.1 nM were performed. Three binding titrations were carried out and the results are averaged and shown in Figure 9. The data were fit with a standard model of one-site ligand binding and gave a KD of 25.7 &#177; 2.1 &#956;M. Evaluation of the binding between VimRod and E-PRD by surface plasmon resonance gave a similar KD value of 19.1 &#177; 1.3 &#956;M4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58537/58537fig1.jpg" /> 
Figure 1: Screen Capture of the Setup of the NMR Experiment. The window shown is used to set up a standard experiment to collect a HSQC dataset. Experiment parameters are read in adjacent to Experiment. The ZGPR experiment shown is chosen as an initial experiment to load the standard and solvent dependent proton parameters. The Title window is used to input experimental details for record keeping purposes. To collect the HSQC spectrum the ZGPR experiment is replaced with SFHMQC3GPPH. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58537/58537fig2.jpg" /> 
Figure 2: Adjusting of NMR Experimental Parameters. The window shown is used for entering the basic parameters for the NMR pulse sequence in order to optimize the signal. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58537/58537fig3.jpg" /> 
Figure 3: NMR Data Processing. Parameters used for processing each of the two dimensions of the NMR spectrum are shown, with arrows indicating those that are typically adjusted. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58537/58537fig4.jpg" /> 
Figure 4: Parameters for NMR Peak Picking. The parameters used for picking NMR peaks in the processed NMR spectrum are shown with typical values. Adjust the ppm range, intensity and number of peaks to optimize the spectra. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58537/58537fig5.jpg" /> 
Figure 5: Representative Peaklist with Intensities. Each peak that is picked in the NMR spectrum is given a number, and its 1H and 15N chemical shifts and signal intensity are displayed. This peaklist can then be used to compare spectra obtained in the presence/absence of an interacting partner. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58537/58537fig6.jpg" /> 
Figure 6: Purification of His6-tagged VimRod by IMAC and S. A. The chromatogram for the elution from the IMAC column shows one major peak of VimRod. B. The chromatogram for the elution from the S column shows one major peak. C. SDS-PAGE of fractions collected over the course of purification: MW standards with the MW indicated in kDa to the left of the gel (M), cell lysate (1), IMAC flow-through (2), wash (3), pooled elution (E1), pooled S elution (E2). Bands visible at higher molecular weights in lanes E1 and E2 are oligomers of pure VimRod as confirmed by western blot (data not shown). <a href="https://www.jove.com/files/ftp_upload/58537/58537fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58537/58537fig7.jpg" /> 
Figure 7: Purification of E-PRD by IMAC and S. A. SDS-PAGE of the IMAC purification showing the molecular weight standards with the MW indicated in kDa to the left of the gel (M) and the eluate from the first IMAC column (E1), the TEV cleavage products (+TEV), and the flow through from the second IMAC column (FT). B. The chromatograph from the S column shows one major peak. C. SDS-PAGE of the molecular weight standards (M) and the fractions from S peak. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58537/58537fig8.jpg" /> 
Figure 8: HSQC Spectra of Wild-type and R1914E Mutant of E-PRD in the Presence and Absence of VimRod. The HSQC spectra show wild-type E-PRD (100 &#181;M) in 20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7 in the absence (A) or presence of 50 &#181;M VimRod (B). Panels C and D are the HSQC spectra of the R1914E mutant (100&#181;M) in the absence or presence of 50 &#181;M VimRod, respectively. In panel E the relative 1H-15N peak intensities of the E-PRD with or without VimRod binding are shown as a function of the peak number, which is arbitrarily assigned and not based on sequence position. These values can be used to define a significance cutoff for peak intensity reduction upon addition of a ligand. If assignments are available, the significant values can often be seen to map to a binding area. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58537/58537fig9.jpg" /> 
Figure 9: Binding of E-PRD to VimRod. E-PRD was diluted in a series of two-fold dilutions from 1.28 mM to 39.1 nM and incubated with labeled VimRod before performing MST analysis. Data from three independent assays were combined. The data were fit to a KD model giving a KD of 25.7 &#956;M with a KD confidence of &#177; 2.1 &#956;M. <a href="https://www.jove.com/files/ftp_upload/58537/58537fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>Sodium phosphate, dibasic (anhydrous)</td>
			<td>6.0 g</td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic (anhydrous)</td>
			<td>3.0 g</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>0.5 g</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>Up to 950 mL</td>
		</tr>
	</tbody>
</table>
Table 1. M9 media for Isotopic Labeling.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>15NH4Cl</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>Glucose (or 13C-glucose)</td>
			<td>2.0 g</td>
		</tr>
		<tr>
			<td>1 M MgSO4</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>50 mM CaCl2</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>20 mg/mL Thiamine</td>
			<td>1.0 mL</td>
		</tr>
		<tr>
			<td>3 mM FeCl3</td>
			<td>400 &#181;L</td>
		</tr>
		<tr>
			<td>Metal Mix (Table 3)</td>
			<td>500 &#181;L</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>Up to 50 mL</td>
		</tr>
	</tbody>
</table>
Table 2. Nutrient Mix for Supplementation of M9 media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>4 mM ZnSO4</td>
			<td>323 mg</td>
		</tr>
		<tr>
			<td>1 mM MnSO4</td>
			<td>75.5 mg</td>
		</tr>
		<tr>
			<td>4.7 mM H3BO3</td>
			<td>145 mg</td>
		</tr>
		<tr>
			<td>0.7 mM CuSO4</td>
			<td>55.9 mg</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>Up to 500 mL</td>
		</tr>
	</tbody>
</table>
Table 3. Metal Mix Supplement for Enriching the MT Nutrient Mix.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>1 mM E-PRD in buffer A1 (&#181;L)</td>
			<td>1mM VimRod in buffer A (&#181;L)</td>
			<td>Buffer A (&#181;L)</td>
			<td>200 &#181;M DSS in D2O (&#181;L)</td>
			<td>Buffer B2 (&#181;L)</td>
			<td>Total Volume (&#181;L)</td>
		</tr>
		<tr>
			<td>E-PRD alone</td>
			<td>50</td>
			<td>0</td>
			<td>50</td>
			<td>50</td>
			<td>350</td>
			<td>500</td>
		</tr>
		<tr>
			<td>E-PRD + VimRod</td>
			<td>50</td>
			<td>50</td>
			<td>0</td>
			<td>50</td>
			<td>350</td>
			<td>500</td>
		</tr>
		<tr>
			<td colspan="2">1Buffer A: 20 mM Tris-HCl, 1 mM DTT, pH 7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">2Buffer B: 23 mM Tris-HCl, 1.14 mM DTT, pH 7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 4. NMR Sample Preparation.

Watch this Scientific Journal Video about Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST at JoVE.com

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

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Interpreting Nuclear Magnetic Resonance Spectra

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12.1K Views.  University of Alberta. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het gebied met eiwitinteractie, zoals of eiwit zich direct bindt aan een klein of een groot molecuul. Het belangrijkste voordeel van deze techniek is dat het eenvoudige en snelle manier is om directe interacties op atomair niveau te detecteren. Zet de luchtstroom aan met het uitwerpcommando ej.Dit brengt het monster van de magneet. Plaats het monster nu in een spinner bovenop de magneet in de opening. Invoegen met d...

Video: Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

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.

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 ...

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The ...

Engineering

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

Advanced NMR Techniques for Glycan-Protein Interaction Analysis

Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.

Author Spotlight: Unveiling the Structural and Dynamic Aspects of Glycan Molecular Recognition

Here, we present a protocol detailing the acquisition, processing, and analysis of a series of NMR experiments aimed at characterizing protein-glycan interactions in solution. Most common ligand-based and protein-based methodologies are outlined, which undoubtedly contribute to the fields of structural glycobiology and molecular recognition studies.

The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and ...

Chemistry

Protocol for NMR 15N Relaxation and hetNOE Experiments in Protein Dynamics Study

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Biology

Microscale Thermophoresis to Study Protein-Lipid Interactions in Solution

Source: Sparks, R. P. et al., <a href="https://app.jove.com/v/60607">Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions.</a>&#160;J. Vis. Exp.&#160;(2022)
This video demonstrates microscale thermophoresis for studying the protein-lipid interaction. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent-labeled proteins in response to a temperature gradient. The fluorescent molecule is mixed with different concentrations of the non-fluorescent lipid molecules, and the mixture of molecules in the solution is loaded into capillaries. A temperature gradient is applied to the samples in the capillaries, and the movement of the fluorescent molecule in the temperature gradient is detected and recorded.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

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