title: Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment.
abstract: Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca(2+) from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca(2+) to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca(2+) binding was investigated by means of a Ca(2+)-sensitive electrode. In all cases, Ca(2+) binds with low affinity; the half-maximum binding constant K(1/2) ranges from 6 to 16 mM. The binding stoichiometry between Ca(2+) ions and carboxylic groups is approximately 1:1; an exception is GARP2, where a binding stoichiometry of approximately 1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca(2+) binding.
authors.affiliation: Institut für Neurowissenschaften und Biophysik, Forschungszentrum Jülich, Jülich, Germany. s.haber-pohlmeier@fz-juelich.de
authors.forename: S
authors.initials: S
authors.lastname: Haber-Pohlmeier
authors.forename: K
authors.initials: K
authors.lastname: Abarca-Heidemann
authors.forename: H G
authors.initials: HG
authors.lastname: Körschen
authors.forename: H Kaur
authors.initials: HK
authors.lastname: Dhiman
authors.forename: J
authors.initials: J
authors.lastname: Heberle
authors.forename: H
authors.initials: H
authors.lastname: Schwalbe
authors.forename: J
authors.initials: J
authors.lastname: Klein-Seetharaman
authors.forename: U B
authors.initials: UB
authors.lastname: Kaupp
authors.forename: A
authors.initials: A
authors.lastname: Pohlmeier
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry*
chemical_list: D009419:Nerve Tissue Proteins; D010455:Peptides; D018698:Glutamic Acid; D002118:Calcium