Conformational changes in calcium-sensor proteins under molecular crowding conditions.

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca(2+) sensors, which undergo conformational changes in response to oscillating intracellular Ca(2+) concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca(2+)-binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase-activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca(2+)-sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca(2+) conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size-exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein-water interface. Our study provides insight into how rather small signaling proteins that have very similar three-dimensional folding patterns differ in their Ca(2+)-occupied functional state under crowding conditions.