title: Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin.
abstract: To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.
authors.affiliation: Department of Molecular Pharmacology, Smith Kline and French Laboratories, Philadelphia, Pennsylvania 19101.
authors.forename: D J
authors.initials: DJ
authors.lastname: Ecker
authors.forename: T R
authors.initials: TR
authors.lastname: Butt
authors.forename: J
authors.initials: J
authors.lastname: Marsh
authors.forename: E J
authors.initials: EJ
authors.lastname: Sternberg
authors.forename: N
authors.initials: N
authors.lastname: Margolis
authors.forename: B P
authors.initials: BP
authors.lastname: Monia
authors.forename: S
authors.initials: S
authors.lastname: Jonnalagadda
authors.forename: M I
authors.initials: MI
authors.lastname: Khan
authors.forename: P L
authors.initials: PL
authors.lastname: Weber
authors.forename: L
authors.initials: L
authors.lastname: Mueller
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D005796:Genes; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014452:Ubiquitins / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D014452:Ubiquitins