title: Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry.
abstract: Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a free-solution label-free method with high sensitivity, to quantify the interaction of neuronal Ca2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase-activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent KD-values of 663 ± 121 nM and 231 ± 63 nM for Ca2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca2+levels.
authors.affiliation: Department of Neuroscience, Biochemistry group, University of Oldenburg, D-26111, Oldenburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Sulmann
authors.affiliation: Department of Chemistry, The Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Kussrow
authors.affiliation: Department of Chemistry, The Vanderbilt Institute for Chemical Biology, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Darryl J
authors.initials: DJ
authors.lastname: Bornhop
authors.affiliation: Department of Neuroscience, Biochemistry group, University of Oldenburg, D-26111, Oldenburg, Germany.
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
mesh_terms: D002118:Calcium / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins / Q000378:metabolism*; D007368:Interferometry; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: C496736:GUCA1A protein, human; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D011956:Receptors, Cell Surface; C110715:guanylate cyclase 1; D006162:Guanylate Cyclase; D002118:Calcium