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

Abstract

Ca(2+)-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca(2+)-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca(2+)-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca(2+) concentration in the 0.1-0.6 μM 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 Ca(2+)-sensor, which appear to be influenced by the presence of free Mg(2+) at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca(2+)-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 Ca(2+)-binding and the conformational transition by competing with Ca(2+), 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.

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

“…Surprisingly, the GCAP proteins do not possess a Ca 2ϩ -myristoyl switch (17,49,50), and the N-terminal myristoyl group remains buried inside the protein in both Ca 2ϩ -free and Ca 2ϩ -bound GCAPs (17,51). Although NMR and crystal structures are known for Ca 2ϩ -bound GCAPs (20 -22, 52), before this study little was known structurally about the Mg 2ϩ -bound/Ca 2ϩ -free activator state of GCAPs and how Ca 2ϩ -induced conformational changes in GCAPs control cyclase activation.…”

A Calcium-Relay Mechanism in Vertebrate Phototransduction

“…. 41 Another important factor involved in sensitivity regulation is the covalent fatty acylation, mainly a myristoylation (as mentioned already above) in GCAP1, which has almost no effect in GCAP2. Presence of the myristoyl group in GCAP1 is essential to shift the Ca 2+ -sensitivity into the physiological range and controls also the affinity of the GCAP1-ROS-GC1 interaction.…”

Conformational Changes in Calcium‐Sensor Proteins under Molecular Crowding Conditions

“…By using the latter approach conformational changes have been detected in enzymes, redox proteins, and Ca 2 + -binding proteins. [11][12][13][14][15][16][17] Furthermore, coupled plasmon-waveguide resonance spectroscopy, a technique related to SPR, has been employed to study conformational changes in G protein-coupled receptors. [18,19] It has been argued that the rather long (ca.…”

“…However, SPR responses obtained with proteins immobilized on dextran-coated sensor chips have been clearly correlated with conformational changes using appropriate controls. [11][12][13][14][15][16][17][18][19] Artefact signals were excluded in a recent study that compares Ca 2+ -induced conformational changes in the neuronal Ca 2 + -sensor protein recoverin with a specifically designed recoverin mutant, which does not undergo a Ca 2+ -triggered conformational change. [16] We noticed that increasing the Ca 2 + concentration ([Ca 2+ ]) into the millimolar range leads to dextran matrix effects on the sensor chip surface independent of the immobilized protein.…”

“…[16] This study was more recently extended by comparing different Ca 2 + -sensor proteins under experimental conditions, in which the influence of ionic strength, divalent cations, and different sensor chip surfaces was tested. [17] This SPR-based approach allows us to investigate Ca 2 + -induced conformational changes in a working range of physiologically relevant free [Ca 2 + ], thereby distinguishing non-specific matrix effects from protein-derived signals in a setting of precisely defined parameters. A further advantage of our method is that it uses a commercial SPR device and a sensor chip, which provides four flow cells to immobilize and investigate up to three proteins and one control surface under identical buffer flow and sample injections.…”