title: Expression, purification and crystallization of a protein resulting from the inversion of the amino-acid sequence of a helical bundle.
abstract: Earlier studies have found that the occurrence of inverse sequence identity in proteins is not indicative of three-dimensional similarity, but rather leads to different folds or unfolded proteins. Short helices, however, frequently keep their conformations when their sequences are inverted. To explore the impact of sequence inversion on long helices, revRM6, with the inverse amino-acid sequence relative to RM6, a highly stable variant of the ColE1 Rop protein, was engineered. RM6 is a highly regular four-α-helical bundle that serves as a model system for protein-folding studies. Here, the crystallization and preliminary crystallographic characterization of revRM6 are reported. The protein was overexpressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belonged to space group P41212, with unit-cell parameters a = b = 44.98, c = 159.74 Å, and diffracted to a resolution of 3.45 Å.
authors.affiliation: Department of Biology, University of Crete, Voutes University Campus, PO Box 2208, 70013 Heraklion, Crete, Greece.
authors.forename: Aikaterini
authors.initials: A
authors.lastname: Kefala
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, Nikolaou Plastira Street 100, 70013 Heraklion, Crete, Greece.
authors.forename: Dina
authors.initials: D
authors.lastname: Kotsifaki
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, Nikolaou Plastira Street 100, 70013 Heraklion, Crete, Greece.
authors.forename: Mary
authors.initials: M
authors.lastname: Providaki
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, Nikolaou Plastira Street 100, 70013 Heraklion, Crete, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Amprazi
authors.affiliation: Department of Biology, University of Crete, Voutes University Campus, PO Box 2208, 70013 Heraklion, Crete, Greece.
authors.forename: Michael
authors.initials: M
authors.lastname: Kokkinidis
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000072756:Protein Conformation, alpha-Helical; D015202:Protein Engineering; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057345:Sequence Inversion; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C054243:Rop protein, ColE1 plasmid
keywords: Rop protein; heptad repeat; hyperthermophilic protein; protein folding; sequence–structure relationship