The NMR solution structure and characterization of pH dependent chemical shifts of the beta-elicitin, cryptogein

The NMR structure of the 98 residue beta-elicitin, cryptogein, which induce s a defence response in tobacco, was determined using N-15 and C-13/N-15 la belled protein samples. Tn aqueous solution conditions in the millimolar ra nge, the protein forms a discrete homodimer where the N-terminal helices of each monomer form an interface. The structure was calculated with 1047 int rasubunit and 40 intersubunit NOE derived distance constraints and 236 dihe dral angle constraints for each subunit using the molecular dynamics progra m DYANA. The twenty best conformers were energy-minimized in OPAL, to give a root-mean-square deviation to the mean structure of 0.82 Angstrom for the backbone atoms and 1.03 Angstrom for all heavy atoms. The monomeric struct ure is nearly identical to the recently derived X-ray crystal structure (ba ckbone rmsd 0.86 Angstrom for residues 2 to 97) and shows five helices, a t wo stranded antiparallel beta-sheet and an Omega-loop. Using H-1, N-15 HSQC spectroscopy the pKa of the N- and C-termini, Tyr(12), Asp(21), Asp(30), A Sp(72), and Tyr(85) were determined and support the proposal of several sta bilizing ionic interactions including a salt bridge between Asp(21) and Lys (62). The hydroxyl hydrogens of Tyr(33) and Ser(78) are clearly observed in dicating that these residues are buried and hydrogen bonded. Two other tyro sines, Tyr(47) and Tyr(87), show pKa's > 12, however, there is no indicatio n that their hydroxyls are hydrogen bonded. Calculations of theoretical pKa 's show general agreement with the experimentally determined values and are similar for both the crystal and solution structures.