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mRNA display selection of a high-affinity, modification-specific phospho-IkappaBalpha-binding fibronectin
C Anders Olson1, Hsiang-I Liao, Ren Sun
1Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125, USA.
ACS Chemical Biology
|July 2, 2008
Summary
Researchers developed a new protein binder, 10C17C25, that specifically targets phosphorylated IkappaBalpha. This tool aids in studying protein modifications and signal transduction pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Post-translational modifications significantly increase proteome complexity.
- Tools for sequence-specific recognition of modified proteins are crucial for research.
- Understanding signal transduction pathways requires real-time visualization methods.
Purpose of the Study:
- To evolve novel, modification-specific ligands targeting phosphorylated IkappaBalpha.
- To develop tools for studying protein trafficking and signal transduction.
- To create sensors for kinase activity.
Main Methods:
- Utilized mRNA display-based in vitro selection with a large protein library.
- Employed fibronectin type III domain-based protein engineering.
- Developed a FRET indicator for kinase activity detection.
Main Results:
- Isolated a fibronectin molecule (10C17C25) binding phospho-IkappaBalpha with high affinity (Kd = 18 nM).
- Demonstrated over 1000-fold specificity for phosphorylated over non-phosphorylated peptides.
- Confirmed specific recognition of endogenous phospho-IkappaBalpha and in vivo stabilization.
- Incorporated the ligand into a FRET sensor for IkappaB kinase (IKK) activity.
Conclusions:
- Novel modification-specific ligands can be evolved using in vitro selection.
- 10C17C25 is a specific and high-affinity binder for phospho-IkappaBalpha.
- Designed adaptors are useful for creating kinase activity sensors and studying signaling.
