The Circular Dichroism Spectroscopy Technique to Study DNA-Protein Interactions
Transcrição
ATP-dependent chromatin remodeling proteins regulate gene expression in tightly-packed chromatin by altering the DNA conformation in an ATP-dependent manner.
To study chromatin remodeler-DNA interactions using circular dichroism, or CD, spectroscopy, take a solution of double-stranded DNA oligonucleotides. Heat to denature the oligonucleotides, and place on ice.
Fast cooling causes the DNA to renature while forming secondary structures. The double-to-single-strand transition region provides an optimal binding site for the chromatin remodelers.
Add the heat-cooled DNA into a cuvette. Add a buffer containing ATP-dependent chromatin remodeler, ATP, and magnesium ions.
The remodeler binds to the DNA, followed by the binding of magnesium ion-complexed ATP to the protein — activating it. The activated remodeler hydrolyzes ATP — inducing a stem-loop conformation in the DNA.
Pass left and right circularly-polarized light — with a 90° phase difference — through the sample. Optically chiral DNA absorbs the left and right circularly-polarized light to different extents.
The differential absorption, termed circular dichroism, results in the transmitted light being elliptically polarized, which is measured as ellipticity. Two positive peaks at specific wavelengths in the CD spectra confirm the stem-loop structure.
Add EDTA to chelate the magnesium ions, inhibiting the ATPase activity.
A change in the CD spectra upon chelation — showing a negative peak and a broad positive peak at specific wavelengths — indicates the disruption of the stem-loop conformation in the absence of ATP hydrolysis.
