Fluorescent-Responsive Synthetic C1b Domains of Protein Kinase C未 as Reporters of Specific High-Affinity Ligand Binding

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• 作者：Nami Ohashi ; Wataru Nomura ; Tetsuo Narumi ; Nancy E. Lewin ; Kyoko Itotani ; Peter M. Blumberg ; Hirokazu Tamamura
• 刊名：Bioconjugate Chemistry
• 出版年：2011
• 出版时间：January 19, 2011
• 年：2011
• 卷：22
• 期：1
• 页码：82-87
• 全文大小：261K
• 年卷期：v.22,no.1(January 19, 2011)
• ISSN：1520-4812

文摘

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 PKC未 C1b domain. These resultant fluorescent-labeled 未C1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent 未C1b domain analogues can be used to evaluate ligand binding affinity.