Biophysial studies of nucleosome structure by circular dichroism, thermal denaturation and ESR spin labeling

Abstract

Rat liver nuclei were digested very briefly by the Ca^+2, Mg^+2 dependent Endogeneous Endonuclease. The digested chromatin was analyzed using exponential sucrose gradients. By plotting the weight average number of subunits released against digestion time, a slowing in the rate of change of chromatin fragments was found in the region of around six subunits. This suggested that the nucleosomes were possibly folded into a chain of discrete superstructures, with 6 to 8 subunits per superbead, and that the DNA between such superbeads was more susceptible to the nuclease digestion than the linker DNA between the nucleosomes. The conformational state of DNA. in the native chromatin and its subunits was analyzed by thermal denaturation and circular dichroism. Monophasic melting profiles were obtained for both the chromatin and its subunits, suggesting that the electrostatic stabilization of the DNA by the histones (HI, H2A, H2B, H3 and H4) was evenly distributed on the chromatin and its subunits. The chromatin and its subunits showed suppressed DNA ellipticities in their circular dichroism spectra. However, upon assembly of the nucleosomes to form a chromatin fiber, the ellipticity increased until the value of chromatin was achieved. We found that at least 8 nucleosomes were required to give the ellipticity of the chromatin, implying that the nucleosomes were possibly folded in an asymmetric fashion. An imidazole spin label (IMDSL) was used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosines in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tyrosines in the nucleosome core particle can be labeled even at 200- to 300-fold molar excess of IMDSL. Conformational changes in the spin labeled histone core and nucleosome core particles due to external perturbations, such as urea, NaCl, pH and temperature, were studied. The nucleosome core was more sensitive than the histone core to urea denaturation. Several conformational transitions in the labeled nucleosome core were observed in the range of 1 mM to 2. 5 M NaCl. A small change was detected at 10 mM NaCl and three major transitions were found between 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl. The labeled nucleosome core particle appeared to be unaffected by changes of pH in the range of 4.5 < pH < 9.5. A thermal denaturation profile, obtained by the ESR method, showed that gradual conformational changes occurred within the inner histone core before the DNA melted. The mode of reconstitution of nucleosome core particles was studied. A mixture of spin labeled histone cores and core DNA was reconstituted by salt step-gradient dialysis. At each step of dialysis, the labeled proteins were examined by ESR. It was found that the histone core bound progressively to the DNA. in the range of 2 M to 0.3 :M NaCl. Full association between histone core and DNA occurred when the ionic strength was less than 0.3 M. The reconstituted nucleosome complexes, purified by isokinetic sucrose gradients, were found to have identical physical properties as the native particle. The role of tyrosines in the reassociation process for the nucleosome core was also investigated. It was found that spin labeling the surface tyrosines on the histone core did not interfere with proper reassociation of the nucleosome core complex. However, when "buried" tyrosines in the histone core were exposed by urea treatment and then spin labeled with IMDSL, additional histone-DNA complexes were formed with properties different from those of native nucleosome cores. This suggests that some of the "buried" tyrosines are essential for the specific histone-histone interactions leading to the histone core structure.