Related Experiment Video
Updated: Jun 13, 2026

Pull-down of Calmodulin-binding Proteins
Published on: January 23, 2012
Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins
Lukas Traxler1, Petr Rathner2, Marc Fahrner1
1Institute of Biophysics, Johannes Kepler University, Gruberstrasse 40, 4020, Linz, Austria.
Abstract:
Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N-termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.
Related Concept Videos
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Assembly of Signaling Complexes
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
MAPK Signaling Cascades
cAMP-dependent Protein Kinase Pathways
Feedback Regulation of Calcium Concentration
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...

