Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3).
Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu(2+), circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu(2+). The NMR spectra indicate that the addition of small amounts of CuSO(4) to 3 (Y1-G2-H3-G4-G5-G6-Y7) causes significant broadening of resonances of the side chain protons (C(beta)H, C(epsilon1)H, and C(delta2)H) of His3 and the side chain C(beta)H of Tyr1 at pH 7.4. In addition, the backbone C(alpha)H resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu(2+) binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers.
Kamiya
Masakatsu
M
Division of Biological Sciences, Graduate School of Science, Hokkaido University, 060-0810, Sapporo, Japan.
Kumaki
Yasuhiro
Y
Nitta
Katsutoshi
K
Ueno
Takafumi
T
Watanabe
Yoshihito
Y
Yamada
Keiko
K
Matsumoto
Takeshi
T
Hikichi
Kunio
K
Matsushima
Norio
N
D000595:Amino Acid Sequence; D027466:Atriplex / Q000235:genetics; D002942:Circular Dichroism / Q000379:methods; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010126:Ozone / Q000494:pharmacology*; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D020449:Repetitive Sequences, Amino Acid
D009842:Oligopeptides; D010940:Plant Proteins; D010126:Ozone; D003300:Copper