title: Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding.
abstract: A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.
authors.affiliation: Centre de Biophysique Moléculaire (CNRS), Orléans, France.
authors.forename: J M
authors.initials: JM
authors.lastname: Bonmatin
authors.forename: J L
authors.initials: JL
authors.lastname: Bonnat
authors.forename: X
authors.initials: X
authors.lastname: Gallet
authors.forename: F
authors.initials: F
authors.lastname: Vovelle
authors.forename: M
authors.initials: M
authors.lastname: Ptak
authors.forename: J M
authors.initials: JM
authors.lastname: Reichhart
authors.forename: J A
authors.initials: JA
authors.lastname: Hoffmann
authors.forename: E
authors.initials: E
authors.lastname: Keppi
authors.forename: M
authors.initials: M
authors.lastname: Legrain
authors.forename: T
authors.initials: T
authors.lastname: Achstetter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023082:Defensins; D006859:Hydrogen; D007301:Insect Hormones / Q000737:chemistry*; D007313:Insecta; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D023082:Defensins; D007301:Insect Hormones; D011994:Recombinant Proteins; C058181:insect defensin A; D006859:Hydrogen