title: Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.
abstract: Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.
authors.affiliation: Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
authors.forename: Nami
authors.initials: N
authors.lastname: Ohashi
authors.forename: Wataru
authors.initials: W
authors.lastname: Nomura
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Narumi
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Lewin
authors.forename: Kyoko
authors.initials: K
authors.lastname: Itotani
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tamamura
mesh_terms: D000595:Amino Acid Sequence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D051745:Protein Kinase C-delta