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.

<ol>
	<li>Vinogradova, O., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+as+a+unique+tool+in+assessment+and+complex+determination+of+weak+protein-protein+interactions.">NMR as a unique tool in assessment and complex determination of weak protein-protein interactions.</a> Topics in Current Chemistry. 326, 35-45 (2012).</li><li>McKercher, M. A., Wuttke, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+Chemical+Shift+Mapping+of+SH2+Peptide+Interactions.">NMR Chemical Shift Mapping of SH2 Peptide Interactions.</a> Methods in Molecular Biology. 1555, 269-290 (2017).</li><li>Al-Jassar, C., Bikker, H., Overduin, M., Chidgey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+Basis+of+Desmosome-Targeted+Diseases.">Mechanistic Basis of Desmosome-Targeted Diseases.</a> Journal of Molecular Biology. 425 (21), 4006-4022 (2013).</li><li>Fogl, C., 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=Mechanism+of+intermediate+filament+recognition+by+plakin+repeat+domains+revealed+by+envoplakin+targeting+of+vimentin.">Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin.</a> Nature Communications. 7, (2016).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Protein Protocols Handbook. , (2002).</li><li>Wishart, D. S., 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=1H,+13C+and+15N+chemical+shift+referencing+in+biomolecular+NMR.">1H, 13C and 15N chemical shift referencing in biomolecular NMR.</a> J. Biomol. NMR. 6, 135-140 (1995).</li><li>Casali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+Host+Strains.">Escherichia coli Host Strains.</a> E. coli Plasmid Vectors. , 27-48 (2003).</li><li>Ericsson, U. B., Hallberg, B. M., DeTitta, G. T., Dekker, N., Nordlund, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermofluor-based+high-throughput+stability+optimization+of+proteins+for+structural+studies.">Thermofluor-based high-throughput stability optimization of proteins for structural studies.</a> Analytical Biochemistry. 357 (2), 289-298 (2006).</li><li>Kozak, S., 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=Optimization+of+protein+samples+for+NMR+using+thermal+shift+assays.">Optimization of protein samples for NMR using thermal shift assays.</a> Journal of Biomolecular Nmr. 64, 281-289 (2016).</li><li>Burgess, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+practical+review+of+size+exclusion+chromatography:+Rules+of+thumb,+limitations,+and+troubleshooting.">A brief practical review of size exclusion chromatography: Rules of thumb, limitations, and troubleshooting.</a> Protein Expression and Purification. , (2018).</li><li>Kunji, E. R. S., Harding, M., Butler, P. J. G., Akamine, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+molecular+mass+and+dimensions+of+membrane+proteins+by+size+exclusion+chromatography.">Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography.</a> Methods. 46 (2), 62-72 (2008).</li><li>Stetefeld, J., McKenna, S. A., Patel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+light+scattering:+a+practical+guide+and+applications+in+biomedical+sciences.">Dynamic light scattering: a practical guide and applications in biomedical sciences.</a> Biophysical Reviews. 8 (4), 409-427 (2016).</li><li>Patel, T. R., Winzor, D. J., Scott, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+ultracentrifugation:+A+versatile+tool+for+the+characterisation+of+macromolecular+complexes+in+solution.">Analytical ultracentrifugation: A versatile tool for the characterisation of macromolecular complexes in solution.</a> Methods. 95, 55-61 (2016).</li><li>Pearson, J. Z., Cole, J. 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=Chapter+One+-+Next-Generation+AUC+Adds+a+Spectral+Dimension:+Development+of+Multiwavelength+Detectors+for+the+Analytical+Ultracentrifuge.">Chapter One - Next-Generation AUC Adds a Spectral Dimension: Development of Multiwavelength Detectors for the Analytical Ultracentrifuge.</a> Methods in Enzymology. 562, 1-26 (2015).</li><li>Gorbet, G. E., Pearson, J. Z., Demeler, A. K., C&#246;lfen, H., Demeler, B., Cole, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+Two+-+Next-Generation+AUC:+Analysis+of+Multiwavelength+Analytical+Ultracentrifugation+Data.">Chapter Two - Next-Generation AUC: Analysis of Multiwavelength Analytical Ultracentrifugation Data.</a> Methods in Enzymology. 562, 27-47 (2015).</li><li>Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-binding+assays+in+biological+liquids+using+microscale+thermophoresis.">Protein-binding assays in biological liquids using microscale thermophoresis.</a> Nature Communications. 1, 100 (2010).</li><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></ol>

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 E. coli</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 (15N, 99%)</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-467</td>
			<td>isotope labelling for NMR</td>
		</tr>
		<tr>
			<td>D2O, 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&#8221; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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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12.0K 조회수.  University of Alberta. 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.

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

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

본질적으로 무질서 단백질의 여러 인산화의 식별을위한 핵 자기 공명 분광학

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

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생화학

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

마이크로 열 영동을 사용하여 ATP에 앱 타머의 결합 부위를 매핑

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

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 ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

곰 팡이의 준비 및 동적인 핵 분극 고체 NMR을 사용 하 여 구조 설명에 대 한 식물 재료

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

네이티브 오토플루임션을 기반으로 sFLIM에 의한 식물 섹션의 형광 프로브와 리그닌 간의 상호 작용 측정

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

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.

단백질-지질 상호작용을 측정하기 위한 마이크로스케일 열영동 사용

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

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

성숙한 쌀의 횡단 단면(오리자 사티 라 L.) 파이펫 팁을 사용하여 전자 현미경 이미징을 스캐닝하기 위한 커널

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

지질 결합 단백질및 알레르겐에서 내인성 리간드의 제거 및 교체

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

최소 샘플이지만 최대 자동화 모드에서 NMR 기반 단편 스크리닝

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

단편 기반 리드 디스커버리에서 스크리닝을 위한 나노 차분 스캔 형광

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...