Dynamics of Conformational Ca2+-Switches in Signaling Networks Detected by a Planar Plasmonic Device

Research output: Contribution to journalArticle

Abstract

Ca2+-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca2+-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca2+-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca2+ concentration in the 0.1-0.6 mu M range. By working at conditions that quantitatively mimic those found in the cell, we show that the method is able to detect subtle differences in the dynamics of each Ca2+-sensor, which appear to be influenced by the presence of free Mg2+ at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca2+-binding constants, directly measured by competition with a chromophoric chelator, and the concerted binding-conformational switch detected by SPR at equilibrium reveals the relative contribution of the conformational change process to the SPR signal. This process appears to be influenced by the presence of other cations that perturb Ca2+-binding and the conformational transition by competing with Ca2+, or by pure electrostatic screening. In conclusion, the approach described here allows a comparative analysis of protein conformational changes occurring under physiologically relevant molecular crowding conditions in ultrathin biosensor layers.

Details

Authors
  • Daniele Dell'Orco
  • Stefan Sulmann
  • Sara Linse
  • Karl-Wilhelm Koch
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Analytical Chemistry
Original languageEnglish
Pages (from-to)2982-2989
JournalAnalytical Chemistry
Volume84
Issue number6
Publication statusPublished - 2012
Publication categoryResearch
Peer-reviewedYes