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Histone H3 thiol reactivity as a probe of nucleosome structure

University of British Columbia

Nucleosomes were prepared from trout testis nuclei by micrococcal nuclease digestion. The reactivity toward N- [ethyl- ³Hlmaleimide (NEM) of the single sulfhydryl group of histone H3 in the nucleosomes was studied under a variety of conditions. Under conditions of low ionic strength, there is negligible reaction of nucleosomes with NEM, suggesting that the cysteinyl residue of H3 is buried. Complete denaturation of nucleosomes in 6 M guanidinium chloride leads to reaction of 2 moles of NEM per mole of nucleosomes, in agreement with the expected presence of 2 moles of H3 per particle. Exposure of nucleosomes to 2 M NaCI or 1 M MgCl₂ leads to exposure of the thiol group. At higher Mg⁺⁺ concentrations, the thiol group remains exposed, but in NaCI solutions, as the salt concentration is increased beyond 2 M, the thiol group returns to an inaccessible state. The reactivity of nucleosome thiol groups is relatively unaffected by urea to approximately 5 M. Between 5 and 8 M urea, a rapid increase in thiol reactivity indicates a cooperative unfolding of the nucleosome core. When added together, urea and salt act in a cooperative manner to expose the H3 sulfhydryl group. Mixtures of oligonucleosomes have also been studied under different conditions. They were found to behave in a similar fashion to monomers in 6 M guanidine, but their thiols react more slowly than those of monomers in high salt. Removal of the amino-terminal regions of the core histones by tryptic digestion has no noticeable effect on the accessibility of nucleosome thiol groups. It is concluded that the carboxy-terminal region of H3 containing Cys 110 is masked mainly by histone-histone interactions in the octameric core complex, and is located in a region which is relatively insensitive to the perturbations induced by trypsin or low concentrations of urea. Nucleosomes reconstituted in the presence of a sulfhydryl reducing agent were indistinguishable from native particles in their reactivity to NEM in low salt buffers, in 2 M NaCl and in 6 M guanidine hydrochloride. These studies indicate that the degree of exposure of H3 sulfhydryl groups in nucleosomes can be effectively monitored using NEM. The carboxy-terminal region of H3 containing Cys 110 seems to be located in a relatively stable region of the nucleosome core, perhaps at the interface between heterotypic tetramers.

Text

2010-03-02

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http://hdl.handle.net/2429/21336

Biochemistry and Molecular Biology

University of British Columbia

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