Ca²⁺ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca²⁺-binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca²⁺ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca²⁺ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca²⁺ sensors is also presented.

Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional coefficient) as the biomolecule. Determination of Rs by SEC has been widely used to monitor conformational changes induced by the binding of calcium (Ca2+) to many Ca2+-sensor proteins. For this class of proteins, SEC separation is based not just on the variation in protein size following Ca2+ binding, but likely arises from changes in the hydration shell structure. This protocol aims to describe a gel filtration experiment on a prepacked column using a Fast Protein Liquid Chromatography (FPLC) system to determine the Rs of proteins with some indications that are specific for Ca2+ sensor proteins.

Calmodulin-like (CML) proteins are major EF-hand–containing, calcium (Ca²⁺)–binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca²⁺ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana. We analyzed Ca²⁺ and Mg²⁺ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca²⁺/Mg²⁺ mixed binding sites and two low-affinity Ca²⁺-specific sites. Binding of Ca²⁺ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca²⁺ or Mg²⁺, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca²⁺-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca²⁺ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca²⁺ pumps.