The extramural grants to Dileep Vasudevan from the Science and Engineering Research Board, Government of India [CRG/2018/000695/PS] and the Department of Biotechnology, Ministry of Science and Technology, Government of India [BT/INF/22/SP33046/2019], as well as the intramural support from the Institute of Life Sciences, Bhubaneswar are greatly acknowledged.&#160;We thank Ms. Sudeshna Sen and Ms. Annapurna Sahoo for their help with histone preparation. The discussions with our colleagues Dr. Chinmayee Mohapatra, Mr. Manas Kumar Jagdev, and Dr. Shaikh Nausad Hossain are also acknowledged.

1. Analytical size-exclusion chromatography to elucidate the oligomeric status and stability of histone chaperones
<ol>
	<li>Analysis of the oligomeric status of histone chaperones
	<ol>
		<li>Equilibrate a 24 mL analytical size-exclusion chromatography (SEC) column with 1.2 column volume (CV), i.e., 28.8 mL of degassed SEC buffer [20 mM of Tris-HCl (pH 7.5), 300 of mM NaCl, and 1 mM of &#946;-mercaptoethanol (&#946;-ME)] at 4 &#176;C (see Table of Materials). 
		NOTE: Column type, buffer composition, and buffer pH may be selected based on the protein of interest. The sample injection volume should not exceed 500 &#181;L for a 24 mL column. Also, the column pressure needs to be maintained below 5 MPa.</li>
		<li>From a higher concentration protein stock solution, prepare 500 &#181;L of 0.5 mg/mL protein sample in degassed SEC buffer and inject it into the pre-equilibrated column using a 500 &#181;L injection loop. Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with the SEC buffer at 4 &#176;C.</li>
		<li>Monitor the elution profile of the protein by measuring absorbance at a wavelength of 280 nm. When dealing with proteins lacking aromatic residues, measure the absorbance at 214 nm.</li>
		<li>Use the elution volume of the protein to calculate its approximate molecular weight in kDa using the standard calibration curve21. 
		NOTE: Calibration curve is prepared by plotting the retention volume of known molecular weight proteins against the log of their respective molecular weights (log Mr), eluted using the same column.</li>
	</ol>
	</li>
	<li>Analysis of the thermal stability of the histone chaperones
	<ol>
		<li>Take 500 &#181;L of 0.5 mg/mL of the protein sample prepared in degassed SEC buffer (same as used in 1.1.1) in individual microcentrifuge tubes and heat each tube to a particular temperature ranging between 20 &#176;C and 90 &#176;C (20 &#176;C, 40 &#176;C, 60 &#176;C, and 90 &#176;C) for 10 min in a water bath.</li>
		<li>Subsequently, centrifuge the heat-treated samples at 16,200 x g for 10 min at 4 &#176;C, collect the supernatant with a micropipette, and inject each sample individually using a 500 &#181;L injection loop into the analytical column, pre-equilibrated with the SEC buffer at 4 &#176;C.</li>
		<li>Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with the SEC buffer at 4 &#176;C.</li>
		<li>Observe the position and height of the elution peaks and look for the appearance of additional peaks for the different samples.</li>
	</ol>
	</li>
	<li>Analysis of the chemical stability of the histone chaperones
	<ol>
		<li>To examine the salt stability of histone chaperones, incubate 500 &#181;L of 0.5 mg/mL protein sample prepared in a Tris buffer [20 mM of Tris-HCl (pH 7.5), and 1 mM of &#946;-ME] supplemented with increasing concentrations of NaCl (300 mM, 600 mM, 1 M, 1.5 M, and 2 M) in separate microcentrifuge tubes for 30 min at 4 &#176;C. Centrifuge the samples at 16,200 x g for 10 min at 4 &#176;C and retain the supernatant.</li>
		<li>Next, load the protein samples in different NaCl concentrations individually, using a 500 &#181;L injection loop into the analytical column pre-equilibrated with 1.2 CV (28.8 mL) of the respective buffer containing increasing NaCl concentrations at 4 &#176;C.</li>
		<li>Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with 1 CV (24 mL) of the respective buffer at 4 &#176;C.</li>
		<li>Observe the position and height of elution peaks and look for the appearance of additional peaks for the different samples.</li>
		<li>Similarly, for urea stability analysis, incubate 500 &#181;L of 0.5 mg/mL protein sample prepared in a Tris buffer [20 mM of Tris-HCl (pH 7.5), and 1 mM of &#946;-ME]&#160;supplemented with increasing urea concentrations (1 M, 2 M, 3 M, 4 M, and 5 M) in separate microcentrifuge tubes for 16 h at room temperature. Centrifuge the samples at 16,200 x g for 10 min at room temperature and retain the supernatant.</li>
		<li>Next, load the urea-treated protein samples individually using a 500 &#181;L injection loop into the analytical column pre-equilibrated with 1.2 CV (28.8 mL) of the corresponding buffer containing different urea concentrations at room temperature.</li>
		<li>Allow the chromatography run to proceed at an isocratic flow rate of 0.2-0.3 mL/min with 1 CV (24 mL) of the respective buffer at room temperature. 
		CAUTION: Do not perform the experiments with buffer containing urea at a lower temperature as urea tends to crystallize and damage the column.</li>
		<li>Observe the position and height of the elution peaks and look for the appearance of additional peaks for the different samples.</li>
	</ol>
	</li>
</ol>
2. Salt gradient-based pull-down assays to understand the type of interactions contributing to the complex formation between histone oligomers and a histone chaperone
<ol>
	<li>For each reaction of pull-down assay, pipette 40 &#181;L of Ni-NTA resin into a spin column and wash with sterile double-distilled water. Subsequently, equilibrate the resin with 100 CV (4 mL) of equilibration buffer [20 mM of Tris-HCl (pH 7.5), 300 mM of NaCl, 10 mM of imidazole, 10 &#181;g/mL of BSA, and 1 mM of &#946;-ME] (see Table of Materials). 
	NOTE: Pull-down can also be performed in a 1.5 mL microcentrifuge tube.</li>
	<li>Prepare the sample by mixing 5 &#181;M of the His-tagged histone chaperone with either 20 &#181;M of histone H2A/H2B dimer or H3/H4 tetramer in the equilibration buffer. Incubate the sample on ice for 1 h. 
	NOTE: H2A/H2B dimer and H3/H4 tetramer are prepared from recombinant human histones21, and the integrity of the oligomers is confirmed based on the estimated molecular masses by analytical ultracentrifugation (AUC). The same histone oligomers have been used for all experiments mentioned below.</li>
	<li>Load the samples into separate pre-equilibrated spin columns with Ni-NTA resin from step 2.1, each labeled for a particular salt concentration, and keep the columns for 30 min at 4 &#176;C. Centrifuge the columns at 1000 x g for 1 min.</li>
	<li>Next, wash the columns with 100 CV (4 mL) of wash buffer [20 mM of Tris-HCl (pH 7.5), 50 mM of imidazole, 0.2% of Tween-20, and 1 mM of &#946;-ME] containing different salt concentrations (i.e., 300 mM,&#160;500 mM, 600 mM, 700 mM, 800 mM, 900 mM and 1 M NaCl). Wash each column with a buffer having a particular salt concentration.</li>
	<li>After the salt washing step, elute the protein from the different columns using 100 &#181;L of elution buffer [20 mM of Tris-HCl (pH 7.5), 300 mM of NaCl, 300 mM of imidazole, and 1 mM of &#946;-ME].</li>
	<li>Subsequently, subject the eluted samples to 18% SDS-PAGE22 and visualize the gel after staining with Coomassie Brilliant Blue R250 (see Table of Materials). Alternatively, you may directly load the resin onto the SDS-PAGE gel instead of eluting the bound protein from the Ni-NTA resin. 
	NOTE: Equilibration, wash, and elution buffer compositions and pH may be modified depending upon the protein of interest.</li>
</ol>
3. Competitive pull-down assay to identify the preference of a histone chaperone for H2A/H2B or H3/H4
<ol>
	<li>Prepare spin column as described in step 2.1</li>
	<li>Incubate 5 &#181;M of the histone chaperone with 20 &#181;M of H2A/H2B dimer in 300 &#181;L of equilibration buffer (prepared in step 2.1) for 30 min on ice. 
	NOTE: The ratio of histone oligomer to histone chaperone in the reaction can be chosen based on known binding stoichiometry data; use five-fold excess histone if no information is available.</li>
	<li>Centrifuge the histone chaperone-H2A/H2B complex at 16,200 x g for 5 min at 4 &#176;C to remove any precipitate. Next, load the sample on the spin column pre-equilibrated with equilibration buffer (prepared in step 2.1) and incubate for 30 min at 4 &#176;C.</li>
	<li>Wash the column with 100 CV (4 mL) of wash buffer [20 mM of Tris-HCl (pH 7.5), 300 mM of NaCl, 50 mM of imidazole, 0.2% of Tween-20, and 1 mM of &#946;-ME] to remove excess H2A/H2B dimer. Next, mix the histone chaperone-H2A/H2B complex with 20-60 &#181;M of H3/H4 tetramer and incubate for 30 min on ice.</li>
	<li>Rewash the column with 100 CV (4 mL) of wash buffer (prepared in step 3.4) to remove any unbound H3/H4 tetramer and elute the sample using elution buffer (prepared in step 2.5). Subject the eluted samples to 18% SDS-PAGE and visualize after staining with Coomassie Brilliant Blue R250. 
	NOTE: The assay could be reversed wherein, first, H3/H4 tetramer can be mixed with the chaperone, the complex allowed to bind to Ni-NTA beads, and the complex then be incubated with varying concentrations of H2A/H2B dimer.</li>
</ol>
4. Analytical ultracentrifugation - sedimentation velocity (AUC-SV) experiments to analyze the binding stoichiometry between histone chaperones and histones
<ol>
	<li>Sample preparation for AUC
	<ol>
		<li>Dialyze the reconstituted histone H2A/H2B dimer, H3/H4 tetramer, and the histone chaperone separately through a 7 kDa cut-off dialysis tubing23, against a dialysis buffer [20 mM of Tris (pH 7.5), 300 mM of NaCl, and 1 mM of &#946;-ME] (see Table of Materials). To minimize background error due to buffer mismatch, perform dialysis extensively against the dialysis buffer, preferably three times over a period of 24 h. 
		NOTE: The initial OD280 of the protein samples should have a two to a three-fold higher value to achieve a final OD280 of 0.3-0.5. This is essentially done to nullify the effects of dilution.</li>
		<li>Purify H2A/H2B dimer, H3/H4 tetramer, and the histone chaperone individually with the dialysis buffer, using analytical size-exclusion chromatography (as mentioned in step 1). Save the buffer from the run to prepare further dilutions later on and to use as a reference in the AUC cell.</li>
	</ol>
	</li>
	<li>Sample loading for AUC
	<ol>
		<li>Mix the purified proteins in a final volume of 450 &#181;L using dialysis buffer from step 4.1.1 to reach an OD280 of 0.3-0.6. Mix the histone chaperone with H2A/H2B dimer or H3/H4 tetramer for complex formation in separate reaction tubes. Incubate the protein mixtures for 2-3 h. 
		NOTE: Alternatively, sedimentation data can be acquired with an interference optical scanning system in the analytical ultracentrifuge. Separately, for mixing purified proteins, fix the histone chaperone concentration and incubate it with increasing concentrations of the histone oligomers to obtain the exact stoichiometry.</li>
		<li>Assemble the cell with a double sector centerpiece and quartz windows for the AUC-SV experiment using an absorbance detector of the analytical ultracentrifuge as described previously in detail24.</li>
		<li>Fill 400 &#181;L of the sample solution and 420 &#181;L of dialysis buffer into the two sectors (sample and reference sectors, respectively) of the cell. 
		NOTE: A larger volume of buffer is used in the reference sector to keep the reference meniscus above the meniscus of the sample. However, while using an optical interference system, fill the two sectors with equal volume.</li>
		<li>Weigh and accurately balance the cells and load them into a four-place titanium rotor (see Table of Materials). Align the cells using the marks provided at the bottom of the cells and the rotor. Load the rotor in the centrifuge, close the lid and allow to develop a vacuum until the pressure drops to less than 15 microns of Hg and the rotor temperature stabilizes to 20 &#176;C (usually takes 2-2.5 h). 
		NOTE: AUC operating parameters include experimental temperature, rotor speed, the interval between scans, and the number of scans to be collected. In the case of SV experiments, the scan interval is given according to the protein&#39;s molecular mass; smaller proteins require larger time intervals between the scans. The rotor speed is also set according to the protein&#39;s expected molecular mass, and the experiment is conducted at 20 &#176;C. The absorbance data is monitored at 280 nm.</li>
		<li>To obtain the exact stoichiometry, keep the histone chaperone concentration constant and incubate with increasing concentrations of histone oligomers to attain saturation.</li>
	</ol>
	</li>
	<li>AUC data analysis
	<ol>
		<li>Perform the data analysis as previously described25. Briefly, calculate the density and viscosity for the buffer components using the program SEDNTERP26 (see Table of Materials). Similarly, calculate the partial specific volume of the protein based on its amino acid composition, also using SEDNTERP.</li>
		<li>Load the data from the AUC machine into the program SEDFIT27 and define the meniscus (red line), the cell bottom (blue line), and data analysis boundaries (green lines). Choose continuous C(s) distribution as a model.</li>
		<li>Next, set resolution maximum up to 100; set sedimentation coefficient (s), s min: 0 and s max: 10-15; set frictional ratio to 1.2 initially and opt to float to derive the ratio from the data; set confidence level (F-ratio; which determines the magnitude of regularization) to 0.68 for 1 sigma regularization; set partial specific volume, buffer density and buffer viscosity values as obtained from SEDNTERP.</li>
		<li>Press RUN to allow the software to solve the Lamm equation27. Adjust the parameters if there is a significant data mismatch. After adjusting the parameters, press FIT to refine all parameters. Assess the quality of fit based on the root-mean-square deviation (RMSD) value, which should be less than 0.01 signal units.</li>
		<li>Estimate the molecular masses of the peaks by choosing the option: show peak &#34;Mw in c(s)&#34; in the display function of the main toolbar, which will provide information about the &#39;s&#39; of the molecule/complex.</li>
	</ol>
	</li>
</ol>
5. Plasmid supercoiling assay to confirm histone chaperoning function
<ol>
	<li>Nucleosome assembly reaction
	<ol>
		<li>Mix 2 &#181;M of H3/H4 tetramer and 4 &#181;M of H2A/H2B dimer with increasing concentrations of the histone chaperone (1-6 &#181;M) in an assembly buffer [20 mM of Tris HCl (pH 7.5), 1 mM of DTT, 1 mM of MgCl2, 0.1 mg/mL of BSA, and 100 mM of NaCl] to a final volume of 50 &#181;L. Incubate the mixture at 4 &#176;C for 30 min.</li>
		<li>Simultaneously, in a separate reaction, pretreat 500 ng of the negatively supercoiled pUC19 plasmid with 1 &#181;g of topoisomerase I enzyme (see Table of Materials) in the assembly buffer in a final volume of 50 &#181;L and incubate at 30 &#176;C for 30 min. 
		NOTE: Topoisomerase I relaxes the supercoiled double-stranded plasmid DNA by generating a single-stranded nick. A topoisomerase I enzyme of eukaryotic origin, such as the commercially available wheat germ topoisomerase I or recombinantly expressed Drosophila melanogaster topoisomerase I, could be used.</li>
		<li>Next, combine the H3/H4 tetramer, H2A/H2B dimer, histone chaperone mixture (from step 5.1.1), the relaxed plasmid DNA reaction mixture (from step 5.1.2), and incubate further at 30 &#176;C for 90 min. 
		NOTE: Set up two control reactions for the assay; one having the histone chaperone and the relaxed plasmid DNA (but not the histones) and the other having the histone oligomers and the relaxed plasmid DNA (but not the histone chaperone).</li>
		<li>Stop the assembly reaction by adding 100 &#181;L of 2x stop buffer (40 mM of EDTA, 2% of SDS, and 0.4 mg/mL of proteinase K) and incubate at 37 &#176;C for 30 min. 
		NOTE: Stop buffer deproteinizes the plasmid DNA by denaturation and proteolysis of bound histones.</li>
	</ol>
	</li>
	<li>Phenol-chloroform extraction and ethanol precipitation
	<ol>
		<li>Add an equal volume of Tris-saturated phenol in the tube containing the reaction mixture from step 5.1.4 and mix well, followed by centrifugation at 16,200 x g for 10 min at room temperature.</li>
		<li>Gently collect the upper aqueous phase having the plasmid DNA with a micropipette and mix with an equal volume of chloroform. Vortex the mixture and centrifuge at 16,200 x g for 10 min at room temperature. 
		NOTE: Isoamyl alcohol could be included at this step to avoid a fuzzy interface between the aqueous and organic phases.</li>
		<li>Next, collect the upper aqueous phase, add 1/10th volume of 3 M sodium acetate (pH 5.5) and 2.5 volumes of ice-cold ethanol (see Table of Materials). Mix the solution well by inverting the tube 3-4 times and keep the mixture in a -20 &#176;C freezer for 30 min for complete precipitation of the plasmid DNA.</li>
		<li>Centrifuge the sample from step 5.2.3 at 16200 x g for 10 min and gently discard the supernatant. Keep the tubes open at room temperature until even trace amounts of ethanol evaporate, leaving the precipitated plasmid DNA in the tube.</li>
	</ol>
	</li>
	<li>Perform the agarose gel electrophoresis to observe the plasmid supercoiling effect.
	<ol>
		<li>Dissolve the precipitated plasmid DNA from step 5.2.4 in sterile double-distilled water.</li>
		<li>Resolve the samples on a 1% agarose gel in 1x Tris-acetate-EDTA (TAE) buffer (40 mM of Tris, 20 mM of acetic acid, and 1 mM of EDTA) (see Table of Materials).</li>
		<li>Stain the gel with 0.2-0.5 &#181;g/mL concentration of ethidium bromide and observe under UV to visualize the DNA bands on the gel.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Electrophoresis. , (2012).</li><li>Andrew, S. M., Titus, J. A., Zumstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dialysis+and+concentration+of+protein+solutions.">Dialysis and concentration of protein solutions.</a> Current Protocols in Toxicology, Appendix 3. , 1-5 (2002).</li><li>Balbo, A., Zhao, H., Brown, P. H., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly,+loading,+and+alignment+of+an+analytical+ultracentrifuge+sample+cell.">Assembly, loading, and alignment of an analytical ultracentrifuge sample cell.</a> Journal of Visualized Experiments. (33), e1530 (2009).</li><li>Padavannil, A., Brautigam, C. A., Chook, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+size+analysis+of+recombinant+importin-histone+complexes+using+analytical+ultracentrifugation.">Molecular size analysis of recombinant importin-histone complexes using analytical ultracentrifugation.</a> Bio-protocol. 10 (10), 3625 (2019).</li><li>Zhao, H., Brautigam, C. A., Ghirlando, R., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+current+methods+in+sedimentation+velocity+and+sedimentation+equilibrium+analytical+ultracentrifugation.">Overview of current methods in sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation.</a> Current Protocols in Protein Science. , (2013).</li><li>Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-distribution+analysis+of+macromolecules+by+sedimentation+velocity+ultracentrifugation+and+Lamm+equation+modelling.">Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modelling.</a> Biophysical Journal. 78 (3), 1606-1619 (2000).</li><li>Kumar, A., Kumar Singh, A., Chandrakant Bob de, R., Vasudevan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+Arabidopsis+thaliana+NAP1-related+protein+2+(AtNRP2)+and+comparison+with+its+homolog+AtNRP1.">Structural characterization of Arabidopsis thaliana NAP1-related protein 2 (AtNRP2) and comparison with its homolog AtNRP1.</a> Molecules. 24 (12), 2258 (2019).</li><li>Liu, W. H., Roemer, S. C., Port, A. M., Churchill, M. E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAF-1-induced+oligomerization+of+histones+H3/H4+and+mutually+exclusive+interactions+with+Asf1+guide+H3/H4+transitions+among+histone+chaperones+and+DNA.">CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.</a> Nucleic Acids Research. 45 (16), 9809 (2017).</li><li>Bowman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+chaperones+Vps75+and+Nap1+form+ring-like,+tetrameric+structures+in+solution.">The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution.</a> Nucleic Acids Research. 42 (9), 6038-6051 (2014).</li><li>Newman, E. R., 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=Large+multimeric+assemblies+of+nucleosome+assembly+protein+and+histones+revealed+by+small-angle+X-ray+scattering+and+electron+microscopy.">Large multimeric assemblies of nucleosome assembly protein and histones revealed by small-angle X-ray scattering and electron microscopy.</a> Journal of Biological Chemistry. 287 (32), 26657-26665 (2012).</li><li>Edlich-Muth, 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=The+pentameric+nucleoplasmin+fold+is+present+in+Drosophila+FKBP39+and+a+large+number+of+chromatin-related+proteins.">The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins.</a> Journal of Molecular Biology. 427 (10), 1949-1963 (2015).</li><li>Franco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+ability+of+nucleoplasmin+to+assemble+and+chaperone+histone+octamers+for+DNA+deposition.">Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition.</a> Scientific Reports. 9 (1), 9487 (2019).</li><li>Xiao, H., Jackson, V., Lei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FK506-binding+protein,+Fpr4,+is+an+acidic+histone+chaperone.">The FK506-binding protein, Fpr4, is an acidic histone chaperone.</a> FEBS Letters. 580 (18), 4357-4364 (2006).</li><li>Graziano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hydrophobic+effect+in+the+salt-induced+dimerization+of+bovine+beta-lactoglobulin+at+pH+3.">Role of hydrophobic effect in the salt-induced dimerization of bovine beta-lactoglobulin at pH 3.</a> Biopolymers. 91 (12), 1182-1188 (2009).</li><li>Burgess, R. J., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+chaperones+in+nucleosome+assembly+and+human+disease.">Histone chaperones in nucleosome assembly and human disease.</a> Nature Structural and Molecular Biology. 20 (1), 14-22 (2013).</li><li>Donham, D. C., Scorgie, J. K., Churchill, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+activity+of+the+histone+chaperone+yeast+Asf1+in+the+assembly+and+disassembly+of+histone+H3/H4-DNA+complexes.">The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4-DNA complexes.</a> Nucleic Acids Research. 39 (13), 5449-5458 (2011).</li><li>Avvakumov, N., Nourani, A., C&#244;t&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+chaperones:+Modulators+of+chromatin+marks.">Histone chaperones: Modulators of chromatin marks.</a> Molecular Cell. 41 (5), 502-514 (2011).</li><li>Ransom, M., Dennehey, B. K., Tyler, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaperoning+histones+during+DNA+replication+and+repair.">Chaperoning histones during DNA replication and repair.</a> Cell. 140 (2), 183-195 (2010).</li><li>Chu, X., 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=Importance+of+electrostatic+interactions+in+the+association+of+intrinsically+disordered+histone+chaperone+Chz1+and+histone+H2A.Z-H2B.">Importance of electrostatic interactions in the association of intrinsically disordered histone chaperone Chz1 and histone H2A.Z-H2B.</a> PLoS Computational Biology. 8 (7), 1002608 (2012).</li><li>Heidarsson, P. O., 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=Disordered+proteins+enable+histone+chaperoning+on+the+nucleosome.">Disordered proteins enable histone chaperoning on the nucleosome.</a> bioRxiv. , (2020).</li></ol>

No conflict of interest was declared.

This work demonstrates and validates a comprehensive set of protocols for the biochemical and biophysical characterization of a putative histone chaperone. Herein, recombinantly expressed and purified NAP family proteins, AtNRP1 and AtNRP2, and the N-terminal nucleoplasmin domain of AtFKBP53 were used to demonstrate the protocols. The same set of experiments could very well be used to delineate the functional attributes of previously uncharacterized histone chaperones from any organism.
The fi...

Nucleosomes composed of DNA and histone proteins form the structural unit of chromatin and regulate several critical cellular events. Nucleosomes are dynamically repositioned and remodeled to make DNA accessible to various processes such as replication, transcription, and translation1,2. Histones that are highly basic either tend to interact with acidic proteins in the cellular milieu or undergo aggregation, thus leading to various cellular defects3,4,5. A group of dedicated proteins called histone chaperones aid the transport of histones from the cytoplasm to the nucleus and prevent aberrant histone-DNA aggregation events6,7. Fundamentally, most histone chaperones store and transfer histones onto DNA at physiological ionic strength, thereby aiding the formation of nucleosomes8,9. Some histone chaperones have a definite preference for the histone oligomers H2A/H2B or H3/H410.
Histone chaperones are characterized based on their ability to assemble nucleosomes dependent or independent of DNA synthesis11. For example, chromatin assembly factor-1 (CAF-1) is dependent while histone regulator A (HIRA) is independent of DNA synthesis12,13. Similarly, the nucleoplasmin family of histone chaperones is involved in sperm chromatin decondensation and nucleosome assembly14. The nucleosome assembly protein (NAP) family members facilitate the formation of nucleosome-like structures in vitro and are involved in the shuttling of histones between cytoplasm and nucleus15. Nucleoplasmins and NAP family proteins are both functional histone chaperones but do not share any structural features. Essentially, no single structural feature allows the classification of a protein as a histone chaperone16. The usage of functional and biophysical assays along with structural studies work best in characterizing histone chaperones.
This work describes biochemical and biophysical methods to characterize a protein as a histone chaperone that aids nucleosome assembly. First, analytical size-exclusion chromatography was carried out to analyze the oligomeric status and stability of histone chaperones. Next, a pull-down assay was performed to determine the driving forces and the competitive nature of histone chaperone-histone interactions. However, the hydrodynamic parameters of these interactions could not be accurately calculated using analytical size-exclusion chromatography because of the protein&#39;s shape and its complexes that impact their migration through the column. Therefore, analytical ultracentrifugation was used, which provides in-solution macromolecular properties that include accurate molecular weight, the stoichiometry of interaction, and the shape of the biological molecules. Past studies have extensively used in vitro histone chaperoning assay to functionally characterize histone chaperones such as yScS11617, DmACF18, ScRTT106p19, HsNPM120. Histone chaperoning assay was also used to functionally characterize the proteins as histone chaperones.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid (glacial)</td>
			<td>Sigma</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>MP Biomedicals</td>
			<td>814326</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>MP Biomedicals</td>
			<td>193983</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA Pure 25M FPLC</td>
			<td>Cytiva</td>
			<td>29018226</td>
			<td>Instrument for protein purification</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>An-60Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>361964</td>
			<td>Rotor for analytical ultracentrifugation</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue R 250</td>
			<td>Sigma</td>
			<td>1.15444</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing (7 kDa cut-off)</td>
			<td>Thermo Fisher</td>
			<td>68700</td>
			<td>For dialysing protein samples</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>MP Biomedicals</td>
			<td>100597</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Loading Dye</td>
			<td>New England Biolabs</td>
			<td>B7025S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>MP Biomedicals</td>
			<td>194822</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Shimadzu</td>
			<td>ATX224R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide (EtBr)</td>
			<td>Sigma</td>
			<td>E8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Doc System</td>
			<td>Bio-Rad</td>
			<td>12009077</td>
			<td>For imaging gels after staining</td>
		</tr>
		<tr>
			<td>Horizontal gel electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td>1704405</td>
			<td>Instrument for agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>MP Biomedicals</td>
			<td>102033</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Eppendorf</td>
			<td>Z683779</td>
			<td>For pipetting of micro-volumes</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN electrophoresis system</td>
			<td>Bio-Rad</td>
			<td>1658000</td>
			<td>Instrument for SDS-PAGE</td>
		</tr>
		<tr>
			<td>N,N-methylene-bis-acrylamide</td>
			<td>MP Biomedicals</td>
			<td>800172</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano drop</td>
			<td>Thermo Fisher</td>
			<td>ND-2000</td>
			<td>For measurement of protein and DNA concentrations</td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Invitrogen</td>
			<td>R901-15</td>
			<td>Resin beads for pull-down assay</td>
		</tr>
		<tr>
			<td>Optima AUC analytical ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>B86437</td>
			<td>Instrument for analytical ultracentrifugation</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>MT30130863</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma</td>
			<td>P4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid isolation kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>1.07393</td>
			<td></td>
		</tr>
		<tr>
			<td>pUC19</td>
			<td>Thermo Fisher</td>
			<td>SD0061</td>
			<td>Plasmid for supercoiling assay</td>
		</tr>
		<tr>
			<td>Refrigerated high-speed centrifuge</td>
			<td>Thermo Fisher</td>
			<td>75002402</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE protein marker</td>
			<td>Bio-Rad</td>
			<td>1610317</td>
			<td></td>
		</tr>
		<tr>
			<td>SEDFIT</td>
			<td></td>
			<td></td>
			<td>Free software program for analytical ultracentrifugation data analysis</td>
		</tr>
		<tr>
			<td>SEDNTERP</td>
			<td></td>
			<td></td>
			<td>Free software program to estimate viscosity and density of buffer and partial specific volume of a protein</td>
		</tr>
		<tr>
			<td>SigmaPrep Spin Columns</td>
			<td>Sigma</td>
			<td>SC1000</td>
			<td>For pull-down assay</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Merck</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>MP Biomedicals</td>
			<td>102918</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL</td>
			<td>Cytiva</td>
			<td>28990944</td>
			<td>Column for analytical size-exclusion chromatography</td>
		</tr>
		<tr>
			<td>Superdex 75 Increase 10/300 GL</td>
			<td>Cytiva</td>
			<td>29148721</td>
			<td>Column for analytical size-exclusion chromatography</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma</td>
			<td>1.10732</td>
			<td></td>
		</tr>
		<tr>
			<td>Topoisomerase I</td>
			<td>Inspiralis</td>
			<td>WGT102</td>
			<td>Enzyme used in plasmid supercoiling assay</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Merck</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>MP Biomedicals</td>
			<td>191450</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>N&#252;ve</td>
			<td>NB 5</td>
			<td>For heat treatment of protein samples</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol (&#946;-ME)</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro characterization of histone chaperones using analytical, pull-down and chaperoning assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

The recombinant N-terminal nucleoplasmin domain of the protein FKBP53 from Arabidopsis thaliana was subjected to analytical SEC. The elution peak volume was plotted against the standard curve to identify its oligomeric state. The analytical SEC results revealed that the domain exists as a pentamer in solution, with an approximate molecular mass of 58 kDa (Figure 1A,B). Further, the nucleoplasmin domain was analyzed for thermal and chemical stability in conjunction w...

Watch this Scientific Journal Video about In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays at JoVE.com

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

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2.6K Views.  Institute of Life Sciences. This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro. 

Video: In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential Scanning Calorimetry &amp;#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent attachment of 76 amino acid ubiquitin modifiers to a target protein, depending on the length and topology of the polyubiquitin chain, can result in different outcomes ranging from protein degradation to changes in the localization and/or activity of modified protein. Three enzymes sequentially catalyze the ubiquitination process: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. E3 ubiquitin ligase determines substrate specificity and, therefore, represents a very interesting study subject. Here we present a comprehensive approach to study the relationship between the enzymatic activity and function of the RING-type E3 ubiquitin ligase. This four-step protocol describes 1) how to generate an E3 ligase deficient mutant through site-directed mutagenesis targeted at the conserved RING domain; 2&#8211;3) how to examine the ubiquitination activity both in vitro and in planta; 4) how to link those biochemical analysis to the biological significance of the tested protein. Generation of an E3 ligase-deficient mutant that still interacts with its substrate but no longer ubiquitinates it for degradation facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, the mutation in the conserved RING domain often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to an RNA-interference approach. Our methods were optimized to investigate the biological role of the plant parasitic nematode effector RHA1B, which hijacks the host ubiquitination system in plant cells to promote parasitism. With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origins.

The goal of this manuscript is to present an outline for the comprehensive biochemical and functional studies of the RING-type E3 ubiquitin ligases. This multistep pipeline, with detailed protocols, validates an enzymatic activity of the tested protein and demonstrates how to link the activity to function.

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...