SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins.
Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.
Wu
Meng
M
Department of Neuroscience and High Throughput Biology Center, School of Medicine, Johns Hopkins University, 733 North Broadway, Baltimore, MD 21205, USA.
Coblitz
Brian
B
Shikano
Sojin
S
Long
Shunyou
S
Cockrell
Lisa M
LM
Fu
Haian
H
Li
Min
M
D048948:14-3-3 Proteins / Q000378:metabolism*; D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015335:Molecular Probes; D009842:Oligopeptides / Q000378:metabolism*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012996:Solutions
D048948:14-3-3 Proteins; D015335:Molecular Probes; D009842:Oligopeptides; D012996:Solutions