Subject(s)
Histones/isolation & purification , Amino Acid Sequence , Animals , Cell Nucleus/ultrastructure , Chickens , Chromatography/methods , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Durapatite , Histones/genetics , Humans , Hydroxyapatites , Indicators and Reagents , Molecular Sequence Data , Nucleoproteins/isolation & purification , Sea Urchins/embryology , Species Specificity , Ultracentrifugation/methodsABSTRACT
The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.
Subject(s)
Trypsin Inhibitors , Trypsin , Animals , Binding Sites , Carboxypeptidases/antagonists & inhibitors , Carboxypeptidases A , Cattle , In Vitro Techniques , Macromolecular Substances , Models, Molecular , Plants/enzymology , Protein Binding , Protein ConformationABSTRACT
A hybrid histone octamer was reconstituted from erythrocyte H2A and H2B, avian [110 Cys-des-thio]histone H3 and the sea-urchin sperm [73Cys]H4 variant. [110Cys-Des-thio]histone H3 was prepared by reaction of natural H3 with Raney nickel. The ability of the hybrid octamer to crystallize to the same form as the natural octamer demonstrated that the chemical modification of cysteine to alanine in H3 and the mutation from threonine to cysteine in sperm H4 do not alter histone-histone interactions in the octamer. Since the sulfhydryl groups of both H4 molecules are fully accessible to 5,5'-dithiobis(2-nitrobenzoate) these residues provide suitable sites for the introduction of a single cysteine-specific label per H4 molecule in the octamer.
Subject(s)
Histones/isolation & purification , Amino Acids/isolation & purification , Animals , Chickens , Erythrocytes/metabolism , Hybridization, Genetic , Male , Sea Urchins , Spermatozoa/metabolismABSTRACT
Histone octamers were covalently labelled with aurothiomalate at amino groups by the method of carbodiimide activation. The labelling procedure was demonstrated to result in the specific covalent coupling through a single bond of the heavy metal atom label to protein amino groups. Such octamers were dissociated to yield soluble H2A-H2B dimers containing three gold atoms per dimer. The dimers were reconstituted with native H3-H4 tetramers to form labelled octamers, which were crystallized to form helical tubes. This strongly suggests that this procedure resulted in minimal changes of protein conformation.
Subject(s)
Gold Sodium Thiomalate , Gold , Histones/metabolism , Affinity Labels , Chromatography, Gel , Crystallization , Electrophoresis, Polyacrylamide Gel , Protein Conformation , SolubilityABSTRACT
Histone octamers were reconstituted from the following preparations: (a) natural histone H3-H4 tetramer and histone H2A-H2B dimer, either selectively extracted from chromatin with solutions chloride or prepared by dissociation of the natural octamer; (b) acid-denatured core histones, either an unfractionated mixture or individually purified proteins. Complexes assembled from these histones elute from exclusion chromatography columns with octamer size as verified by cross-linking with dimethylsuberimidate. The reconstituted octamers all crystallize in the same form of helical tubes as the natural octamer.
