A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure.
PCaP1, a hydrophilic cation-binding protein, is bound to the plasma membrane in Arabidopsis thaliana. We focused on the physicochemical properties of PCaP1 to understand its uniqueness in terms of structure and binding of metal ions. On fluorescence analysis, PCaP1 showed a signal of structural change in the presence of Cu(2+). The near-UV CD spectra showed a marked change of PCaP1 in CuCl(2) solution. The far-UV CD spectra showed the presence of alpha-helices and the intrinsically unstructured region. However, addition of Cu(2+) gave no change in the far-UV CD spectra. These results indicate that Cu(2+) induced a change in the tertiary structure without changing the secondary structure. The protein was sensitive to proteinase in the presence of Cu(2+), supporting that Cu(2+) is involved in the structural change. The PCaP1 solution was titrated with CuCl(2) and the change in the fluorescence spectrum was monitored to characterize Cu(2+)-binding properties. The obtained values of K(d) for Cu(2+) and the ligand-binding number were 10 microM and six ions per molecule, respectively. These findings indicate that PCaP1 has a high Cu(2+)-binding capacity with a relatively high affinity. PCaP1 lacks cysteine and histidine residues. A large number of glutamate residues may be involved in the Cu(2+) binding.
Nagasaki-Takeuchi
Nahoko
N
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Miyano
Masashi
M
Maeshima
Masayoshi
M
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; C521040:PCaP1 protein, Arabidopsis; D011994:Recombinant Proteins; D003300:Copper