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

Herling, Therese W.; O'Connell, David J.; Bauer, Mikael C. LU ; Persson, Jonas; Weininger, Ulrich LU ; Knowles, Tuomas P J and Linse, Sara LU (2016) In Biophysical Journal 110(9). p.1957-1966
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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biophysical Journal
volume
110
issue
9
pages
10 pages
publisher
Rockefeller University Press
external identifiers
  • scopus:84966365345
  • wos:000375896400006
ISSN
0006-3495
DOI
10.1016/j.bpj.2016.03.038
language
English
LU publication?
yes
id
f725e60f-a3cf-4e36-95d9-8a975ec5e8f0
date added to LUP
2016-09-28 11:11:11
date last changed
2017-05-21 04:54:05
@article{f725e60f-a3cf-4e36-95d9-8a975ec5e8f0,
  abstract     = {<p>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.</p>},
  author       = {Herling, Therese W. and O'Connell, David J. and Bauer, Mikael C. and Persson, Jonas and Weininger, Ulrich and Knowles, Tuomas P J and Linse, Sara},
  issn         = {0006-3495},
  language     = {eng},
  month        = {05},
  number       = {9},
  pages        = {1957--1966},
  publisher    = {Rockefeller University Press},
  series       = {Biophysical Journal},
  title        = {A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions},
  url          = {http://dx.doi.org/10.1016/j.bpj.2016.03.038},
  volume       = {110},
  year         = {2016},
}