Our protocol describes how techniques like pull-down assays analytical size exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used to confirm whether a protein is a histone chaperone. The techniques covered here, when using tandem, could be applied directly to characterize a putative chaperone. his could go along with the structural biology technique.
The techniques are applicable to the field of chromatin research. However, the individual methods could be applied to any other protein of interest, as required. Demonstrating the procedure will be Mr.Surajit Gandhi, a PhD student from my laboratory, for the complicated pull-down assay;Ms.
Archana Samal, a PhD student, along with Mr.Ruchir C.Bobde for the analytical ultra-centrifugation experiment;Mr. Somanath Baral, a PhD student;along with Mr.Ketul Saharan for the plasmid super-coiling assay First, mix five-micromolar histone chaperones with 20-micromolar H2A/H2B dimers in 300 microliters of equilibration buffer. Centrifuge the histone chaperone H2A/H2B complex to remove any precipitate.
Load the sample on the spin column pre-equilibrated with equilibration buffer. Wash the column with 100 column volumes or four milliliters of wash buffer to remove excess H2A/H2B dimer. Next, mix the histone chaperone H2A/H2B complex with 20-to 60-micromolar H3/H4 tetramer.
Rewash the column with 100 column volumes or four milliliters of wash buffer to remove any unbound H3/H4 tetramer, then elute the samples with elution buffer. Subject the eluted samples to 18%SDS-PAGE. Then, stain with Coomassie brilliant blue R250 and visualize the gel.
Purify the H2A/H2B dimer, H3/H4 tetramer in the histone chaperone individually with the dialysis buffer using analytical size exclusion chromatography. Save the buffer from the run to prepare further dilutions later on and to use as a reference in the AUC cell. For AUC samples, mix the purified chaperones with dimer or tetramer in separate tubes to a final volume of 450 microliters using the saved dialysis buffer to reach an OD280 of 0.3 to 0.6.
Assemble the cell with a double-sector centerpiece and quartz windows provided with an analytical ultra centrifuge with an absorbance detector. Fill 400 microliters of the sample solution and 420 microliters of dialysis buffer into the reference and sample sectors of the cell, respectively. Weigh and accurately balance the cells and load them into a four-hole titanium rotor.
Align the cells using the marks at the bottom of the cells and the rotor. Load the rotor in the centrifuge, close the lid, and start the run, which allows a vacuum to develop and the temperature to stabilize. For data analysis, calculate the density and viscosity for the proteins buffer components and partial specific volume based on the amino acid composition of the protein using the program SEDNTERP.
Load the data from the AUC machine into the program SEDFIT. Define the meniscus, red line, the cell bottom, blue line, and data analysis boundaries green lines. Choose Continuous CS distribution as a model.
In the parameters section, set resolution maximum up to 100. Next, set sedimentation coefficient minimum to zero and maximum to 10 to 15. Set the frictional ratio to 1.2 initially and opt to float to derive the ratio from the data.
Set the F-ratio to 0.68 for one sigma regularization. Set partial specific volume, buffer density, and viscosity as obtained from the SEDNTURP. Press Run to allow the software to solve the lam equation.
Press Fit to refine. Choose Show Peak Mw in CS in the display function of the main toolbar to estimate molecular masses. Mix two-micromolar H3/H4 tetramer and four-micromolar H2A/H2B dimer with increasing concentrations of histone chaperone, ranging from one-to six-micromolar, in an assembly buffer to a final volume of 50 microliters.
Incubate the mixture at four degrees Celsius for 30 minutes. Simultaneously, pretreat 500 nanograms of the negatively super-coiled PUC-19 plasmid with one microgram of topoisomerase I enzyme in the assembly buffer in a final volume at 50 microliters. Next, combine the pre-mixed solution containing tetramer, dimer, and histone chaperone with the relaxed plasma DNA.
Add 100 microliters of 2X stop buffer and incubate to end the assembly reaction by deproteinizing the plasmid DNA. Add an equal volume of Tris-saturated phenol in the tube containing the reaction mixture and mix the contents of the reaction mixture well. Then, centrifuge the sample.
Gently collect the upper aqueous phase containing the plasmid DNA with a micropipette and mix an equal volume of chloroform. Vortex the mixture. Next, collect the upper aqueous phase, add a 1/10 volume of three-molar sodium acetate of pH 5.5, and 2.5 volumes of ice-cold ethanol.
Mix the solution well by inverting the tube three to four times. Centrifuge the sample at 16, 200 G for 10 minutes 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.
The analytical SEC results revealed that the recombinant N-terminal nucleoplasmin domain of the Arabidopsis thaliana protein FKBP53 is a pentamer of 65 kilodaltons in solution. The nucleoplasmin sample subjected to heat treatment showed that the domain is highly thermo-stable. The nucleoplasmin domain displayed salt stability up to two molars of sodium chloride and urea stability up to four molars.
A pull-down assay revealed that the interaction of the nucleoplasmin domain with H2A/H2B dimer was stable up to 0.4-molar sodium chloride, whereas the association with H3/H4 was stable up to 0.7 molars. The chaperone binds H2A/H2B dimer and H3/H4 tetramer simultaneously, irrespective of the order in which they are added to the chaperone, indicating separate interaction sites for the oligomers. The estimated molecular masses through AUC-SV reveal a 1:1 stoichiometry of nucleoplasmin domain with the histone oligomers.
In the plasmid super-coiling assay, recombinant plant histone chaperones AtNRP1 and AtNRP2 increased the amount of super-coiled plasmid, suggesting it could deposit histones onto the DNA to form nucleosomes, causing DNA super coiling. Plasmid super-coiling assay needs special care and possible optimization for histone DNA and chaperone characterization. Isomeric characterization telemetry can be used as a follow-up to analytical size-exclusion experiments to confirm the binding stability.
