Design of a hybrid biosensor for enhanced phosphopeptide recognition based on a phosphoprotein binding domain coupled with a fluorescent chemosensor.
Protein-based fluorescent biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated biological systems. Here, we present the design of a hybrid biosensor, specific for a bis-phosphorylated peptide, based on a natural phosphoprotein binding domain coupled with an artificial fluorescent chemosensor. The hybrid biosensor consists of a phosphoprotein binding domain, the WW domain, into which has been introduced a fluorescent stilbazole having Zn(II)-dipicolylamine (Dpa) as a phosphate binding motif. It showed strong binding affinity and high sensing selectivity toward a specific bis-phosphorylated peptide in the presence of various phosphate species such as the monophosphorylated peptide, ATP, and others. Detailed fluorescence titration experiments clearly indicate that the binding-induced fluorescence enhancement and the sensing selectivity were achieved by the cooperative action of both binding sites of the hybrid biosensor, i.e., the WW domain and the Zn(II)-Dpa chemosensor unit. Thus, it is clear that the tethered Zn(II)-Dpa-stilbazole unit operated not only as a fluorescence signal transducer, but also as a sub-binding site in the hybrid biosensor. Taking advantage of its selective sensing property, the hybrid biosensor was successfully applied to real-time and label-free fluorescence monitoring of a protein kinase-catalyzed phosphorylation.
Anai
Takahiro
T
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan.
Nakata
Eiji
E
Koshi
Yoichiro
Y
Ojida
Akio
A
Hamachi
Itaru
I
D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000138:chemical synthesis / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D010748:Phosphopeptides; D010750:Phosphoproteins