Characterization of the basic amphiphilic alpha-helix calmodulin-binding domain of a 61.5 kDa tobacco calmodulin-binding protein.
A 19-amino acid residue peptide, Gly-Trp-Leu-Lys-Ile-Lys-Ala-Ala-Met-Arg-Trp-Gly-Phe-Phe-Val-Arg-Lys-Lys- Ala, corresponding to the basic amphiphilic alpha-helix (BAA) motif at the C-terminus of a recombinant tobacco calmodulin-binding protein, TCB60, was synthesized. The interaction of the synthetic binding domain with calmodulin (CaM) was analyzed by gel mobility shift assays, phosphodiesterase competition assays, and fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy. Mobility shift assays showed an apparent 2 kDa increase in CaM Mr in presence of synthetic peptide and CaCl2 in 4 M urea polyacrylamide gel electrophoresis. HPLC measurements of hydrolysis of cyclic AMP by CaM-dependent phosphodiesterase indicated the synthetic peptide competitively inhibits (Ki = 15-20 nM) stimulation of phosphodiesterase activity by CaM. Upon binding CaM, the fluorescence emission maximum of the synthetic peptide, which contained two tryptophanyl residues, shifted toward blue and increased in intensity. The circular dichroism spectra indicated the ellipticity of CaM increased at 208 and 222 nm upon complex formation with the synthetic peptide. 1H NMR studies showed that the peptide interacts with the aromatic residues in domains I and III of CaM. Taken together, these data provide direct evidence that the structurally conserved basic amphiphilic alpha-helix CaM-binding domain of the recombinant tobacco CaM-binding protein interacts with CaM at physiologically significant nanomolar concentrations and the microenvironments of both CaM and the synthetic binding domain are modified upon complex formation.
Dash
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Department of Plant Molecular Physiology, University of Hawaii, Honolulu 96822, USA.
Niemaczura
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Harrington
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HM
D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D010947:Plants, Toxic; D011485:Protein Binding; D017510:Protein Folding; D014026:Nicotiana / Q000737:chemistry*
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010940:Plant Proteins