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

Research output: Contribution to journalArticle

Abstract

The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.

Details

Authors
  • Therese W. Herling
  • David J. O'Connell
  • Mikael C. Bauer
  • Jonas Persson
  • Ulrich Weininger
  • Tuomas P J Knowles
  • Sara Linse
Organisations
External organisations
  • University of Cambridge
  • University College Dublin
  • Uppsala University
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biophysics
Original languageEnglish
Pages (from-to)1957-1966
Number of pages10
JournalBiophysical Journal
Volume110
Issue number9
Publication statusPublished - 2016 May 10
Publication categoryResearch
Peer-reviewedYes