title: Conformational changes in the nucleosome followed by the selective accessibility of histone glutamines in the transglutaminase reaction: effects of ionic strength.
abstract: Transglutaminases, the enzymes that catalyze the acyl-transfer reaction between glutamine and primary amines, have been used to introduce probes into proteins in order to perform structural studies using physical techniques. Here we use an original approach in which the increasing accessibility of the glutamines of core histones to TGase is used to monitor the salt-induced conformational changes of the nucleosome. The rationale of this strategy is that the accessibility of a glutamine to transglutaminase depends on the blockage due to the presence of either other histones or DNA. At low ionic strength, only glutamines on the N-terminal tails of H2B and H3 are labeled with monodansylcadaverine when core particles are incubated with transglutaminase. The partial unfolding that occurs when going to higher ionic strength values results in an increase in the number of reactive glutamines up to a maximum value of 16 per nucleosome. Labeling of some residues (e.g., Gln(104) and Gln(112) of H2A) requires the unwinding of DNA and the dissociation of the H2A--H2B dimers. Gln(76) of H3 is labeled in the H3--H4 tetramer only when the H2A--H2B dimers are dissociated. Interestingly, the labeling of Gln(95) of H2B exclusively depends on the unwinding of DNA. The accurate analysis of these results indicates that the ionic-dependent unwinding of the DNA may occur following a two-state model.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Valencia, E-46100 Burjassot, Valencia, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Ballestar
authors.forename: M
authors.initials: M
authors.lastname: Boix-Chornet
authors.forename: L
authors.initials: L
authors.lastname: Franco
mesh_terms: D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000378:metabolism; D002384:Catalysis; D002645:Chickens; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D005973:Glutamine / Q000737:chemistry* / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D009707:Nucleosomes / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism*; D009994:Osmolar Concentration; D011487:Protein Conformation; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006657:Histones; D046911:Macromolecular Substances; D009707:Nucleosomes; D005973:Glutamine; D004247:DNA; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine