کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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1174613 | 961761 | 2010 | 6 صفحه PDF | دانلود رایگان |

A fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen fragment-size compounds that weakly and slowly inhibit protein–peptide interactions was established. The interactions were detected by the increased diffusion time of a fluorescently labeled peptide probe after binding to its interacting protein. We analyzed the interactions between the c-Cbl TKB domain and phosphopeptides derived from ZAP-70, APS, and EGFR with the FCS assay and obtained 6 hit fragments that bound to the c-Cbl interaction sites. The binding amounts of the fragments were measured by direct binding measurements using surface plasmon resonance, and 5 fragments were found to bind selectively. The effect of 2 of the 5 fragments on the interaction with c-Cbl and the peptide exhibited strong time dependency. Furthermore, the inhibition by the selected 5 fragments on the protein–peptide interaction was confirmed by their effect on pull-down assays of c-Cbl with the biotin-conjugated interaction peptides. These results indicate the advantage of our FCS-based assay to study the time-dependent binding of compounds to their target protein.
Journal: Analytical Biochemistry - Volume 402, Issue 1, 1 July 2010, Pages 26–31