title: The structure of chromatin reconstituted with phosphorylated H1. Circular dichroism and thermal denaturation studies.
abstract: The effect of binding enzymatically phosphorylated H1 histone on the structure of reconstituted chromatin was determined by circular dichroism and thermal denaturation studies at low ionic strength. Procedures were developed for the selective removal of the lysine-rich histones H1 and H5 from chicken erythrocyte chromatin (stripped chromatin) and for the specific reconstitution of chromatin, without nucleosome sliding. Reconstitution was carried out with either rat thymus H1, phosphorylated rat thymus H1 (containing an average of 5.3 phosphates/molecule, or chicken erythrocyte H5. The maximum ellipticity in 1.0 mM phosphate buffer, pH 7.4, of stripped chromatin [( theta]282.5 = 4800 degree cm2/dmol) was effectively reduced to the value for native chromatin [( theta]282.5 = 3900 degree cm2/dmol) through reconstitution with approximately one molecule of either H1 [( theta]282.5 = 4050 degree cm2/dmol) or phosphorylated H1 [( theta]282.5 = 4100 degree cm2/dmol) per 200 base pairs of DNA. As the circular dichroic spectra for chromatin reconstituted with H1 and phosphorylated H1 are similar, it appears that phosphorylation per se does not induce a major structural alteration of chromatin structure. Thermal denaturation studies at low ionic strength of chromatin reconstituted with either H1 or phosphorylated H1 revealed a transition that occurs at a slightly lower temperature than the high temperature transition of native chromatin. This transition was shifted approximately 1 degree C to lower temperature when the chromatin was reconstituted with phosphorylated H1 as compared to H1. Thus, phosphorylation of H1 caused a significant destabilization of the chromatin structure at low ionic strength. When thermal denaturation was carried out on chromatin reconstituted with H5, native-like profiles were obtained.
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure*; D002942:Circular Dichroism; D004912:Erythrocytes / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D009994:Osmolar Concentration; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation
chemical_list: D002843:Chromatin; D006657:Histones