A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Cellular signaling relies on protein-ligand binding, but equilibrium parameters are difficult to measure in solution.
• Existing methods often require immobilization or viscous matrices, limiting applicability.
• Accurate characterization of binding events is crucial for understanding biological regulation.

Purpose of the Study:

• To develop a microfluidic platform for quantifying protein-ligand binding equilibria in native solution conditions.
• To characterize the interaction between calmodulin and creatine kinase, identifying creatine kinase as a novel calmodulin target.
• To investigate the calcium ion dependence of calmodulin's ligand-binding affinities and its effect on creatine kinase activity.

Main Methods:

• Development of a microfluidic assay for real-time detection of protein-ligand interactions.
• Measurement of absolute electrophoretic mobilities of solvated proteins.
• Quantitative analysis of binding affinities and kinetic parameters under varying solution conditions.

Main Results:

• Demonstrated a microfluidic platform with second-timescale assay capability, eliminating immobilization requirements.
• Quantified absolute electrophoretic mobilities for solvated proteins and compared results across different solution conditions.
• Identified creatine kinase as a novel calmodulin target and characterized the calcium-dependent binding affinities.
• Observed increased creatine kinase activity upon interaction with calmodulin.

Conclusions:

• Microfluidic techniques offer a powerful tool for characterizing biomolecular interactions under native solution conditions.
• The developed platform enables precise quantification of binding equilibria and affinities.
• The findings provide new insights into calcium-mediated cellular signaling and calmodulin's regulatory roles.