Powerful Binders for the D‑Dimer by Conjugation of the GPRP Peptide to Polypeptides from a Designed SetIllustrating a General Route to New Binders for Proteins
journal contributionposted on 2013-01-16, 00:00 authored by Ramesh Ramapanicker, Xiaojiao Sun, Johan Viljanen, Lars Baltzer
The synthetic tetrapeptide GPRP based on the amino-terminal GPR sequence of the fibrin α-chain binds the D-dimer protein with a dissociation constant KD of 25 μM. The D-dimer protein, a well-known biomarker for thrombosis, contains two cross-linked D fragments from the fibrinogen protein formed upon degradation of the fibrin gel, the core component of blood clots. In order to develop a specific high-affinity binder for the D-dimer protein, GPRP was conjugated via an aliphatic spacer to each member of a set of sixteen polypeptides designed for the development of binder molecules for proteins in general. The binders were individually characterized and ranked using surface plasmon resonance (SPR) analysis. The dissociation constant of the complex formed from the D-dimer and 4-D15L8-GPRP labeled with fluorescein was determined by fluorescense titration and found to be 3 nM, an affinity 4 orders of magnitude higher than that of free GPRP. According to SPR analysis, binding was completely inhibited by free GPRP at mM concentrations and the polypeptide conjugate was therefore shown to bind specifically to the binding site of GPRP. Affinities were further enhanced by dimerization of the polypeptide conjugates via a bifunctional linker resulting in dissociation constants that were further decreased (affinities increased) by factors of 2–4. The results suggest an efficient route to specific binders for proteins based on short peptides with affinities that need only to be modest, thus shortening the time of binder development dramatically.
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General Routepolypeptide conjugatefibrinogen proteinbifunctional linkerbinder molecules25 μ Mpolypeptide conjugatesmM concentrationsPowerful Bindersfluorescense titrationbinding sitecore componenttetrapeptide GPRP3 nMbinder developmentKDaffinity 4 orderssurface plasmon resonanceSPR analysisaliphatic spacerblood clotsNew Bindersfibrin geldissociation constantsGPRP Peptide15L