Calcium-Induced Protein Folding in Calumenin and Calmodulin

Abstract

Binding of calcium — and small molecules in general — often induce conformational changes in large molecules and complexes. The degree and type of change varies, but the resulting shift in specific affinities ultimately induces a physiological response. It is therefore important for our understanding of responses at the cellular level to define coupled changes at the molecular level.

Calumenin, a six-EF-hand calcium-binding protein localized in the endoplasmic reticulum, undergoes substantial calcium-induced rearrangement. We have demonstrated how calumenin changes from being unfolded in the absence of calcium to a compact trilobal fold in the presence of calcium (Mazzorana et al., PLoS One 11:e0151547, 2016).

Here, we describe protocols for the expression and purification of calumenin and calmodulin, another EF-hand protein modulated by calcium, along with protocols for biophysical techniques used to characterize calcium-induced changes to protein conformation. Analytical size-exclusion chromatography in the presence and absence of calcium provides an informed indication of any larger conformational movements. Circular dichroism spectroscopy reveals alterations to the secondary or tertiary structure, while small-angle X-ray scattering explores changes further providing low-resolution conformational details.

Surface plasmon resonance estimates binding kinetics and affinities completing the biophysical description of these events.