Resonance assignment, cysteine-pairing elucidation and secondary-structure determination of capsicein, an alpha-elicitin, by three-dimensional 1H NMR.
Difficulties encountered in the interpretation of two-dimensional NMR spectra of proteins exceeding roughly 100 amino acids, including resonance overlap and line broadening due to longer correlation times and/or aggregation phenomena, can be overcome by using three-dimensional 1H-NMR experiments. The improvement of spectral resolution using these experiments allows the size of molecules amenable to structure determination by NMR spectroscopy to be extended. A three-dimensional non-selective homonuclear Hartmann-Hahn/nuclear Overhauser effect spectroscopy experiment was performed on capsicein, a 10161-Da elicitin secreted by the Phytophthora capsici fungus. Sequential assignment and secondary structure determination is illustrated for beta-sheet, alpha-helix and loop structures by analysis of planar cross sections perpendicular to the omega 2 or omega 3 axis at the amide proton resonance frequencies. Cysteine pairing was established in the course of the investigation. The secondary structure topology of the molecule is composed of five helices and an antiparallel beta-sheet. Four of the helices compose two pairs running antiparallel while the last one is parallel to the beta-sheet.
Bouaziz
S
S
Laboratoire de Résonance Magnétique Nucléaire, Institut de Chimie des Substances Naturelles du CNRS, Gif-sur-Yvette, France.
van Heijenoort
C
C
Guittet
E
E
Huet
J C
JC
Pernollet
J C
JC
D000595:Amino Acid Sequence; D003545:Cysteine; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D010838:Phytophthora; D017433:Protein Structure, Secondary
D005656:Fungal Proteins; C061033:Alpha-elicitin capsicein protein, Phytophthora capsici; D006859:Hydrogen; D003545:Cysteine