Authors: Therese W. Herling, David J. O’Connell, Mikael C. Bauer, Jonas Persson, Ulrich Weininger, Tuomas P. J. Knowles, and Sara Linse
Biophys J. 2016, 110(9), 1957–1966. DOI: 10.1016%2Fj.bpj.2016.03.038
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, they 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, they obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions.
They apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, they explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. They 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.
In this paper, Herling et al. 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. They do this using a microfluidic free-flow electrophoresis platform. In this manner, they are able to determine the dissociation constant of calmodulin with a novel binding protein—creatine kinase, and look at how calcium concentration alters this.
The authors compare protein binding affinity measurements in free solution (obtained using free flow electrophoresis and thermophoresis), and on a surface (obtained using SPR), noting that SPR measurements report a considerably lower KD relative to the in solution methods, which correlate well.
Therapeutic area: protein-protein interactions