title: NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling.
abstract: NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successful overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.
authors.affiliation: Department of Pharmacy, University of Patras, GR-26504 Patras, Greece.
authors.forename: Dionysios J
authors.initials: DJ
authors.lastname: Vourtsis
authors.affiliation: Department of Pharmacy, University of Patras, GR-26504 Patras, Greece.
authors.forename: Christos T
authors.initials: CT
authors.lastname: Chasapis
authors.affiliation: Department of Pharmacy, University of Patras, GR-26504 Patras, Greece.
authors.forename: George
authors.initials: G
authors.lastname: Pairas
authors.affiliation: Institute of Physiology II, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Detlef
authors.initials: D
authors.lastname: Bentrop
authors.affiliation: Department of Pharmacy, University of Patras, GR-26504 Patras, Greece. Electronic address: G.A.Spyroulias@upatras.gr.
authors.forename: Georgios A
authors.initials: GA
authors.lastname: Spyroulias
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000648:ultrastructure*; D001427:Bacterial Toxins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000596:Amino Acids; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin
keywords: Anthrax Lethal Factor; NMR spectroscopy; Protein expression; Selective labeling strategies