Resonance assignment, cysteine‐pairing elucidation and secondary‐structure determination of capsicein, an α‐elicitin, by three‐dimensional 1H NMR

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Abstract
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 β‐sheet, α‐helix and loop structures by analysis of planar cross sections perpendicular to the ω2 or ω3 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 β‐sheet. Four of the helices compose two pairs running antiparallel while the last one is parallel to the β‐sheet.