title: A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions.
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.
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Therese W
authors.initials: TW
authors.lastname: Herling
authors.affiliation: School of Biomolecular and Biomedical Science, University of College Dublin, Dublin, Ireland.
authors.forename: David J
authors.initials: DJ
authors.lastname: O'Connell
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden.
authors.forename: Mikael C
authors.initials: MC
authors.lastname: Bauer
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden; Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
authors.forename: Jonas
authors.initials: J
authors.lastname: Persson
authors.affiliation: Department of Biophysical Chemistry, Lund University, Lund, Sweden.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Weininger
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. Electronic address: tpjk2@cam.ac.uk.
authors.forename: Tuomas P J
authors.initials: TP
authors.lastname: Knowles
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, Lund, Sweden. Electronic address: sara.linse@biochemistry.lu.se.
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
mesh_terms: D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D003402:Creatine Kinase / Q000378:metabolism*; D004586:Electrophoresis; D008024:Ligands; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013997:Time Factors
chemical_list: D002147:Calmodulin; D008024:Ligands; D003402:Creatine Kinase; D002118:Calcium