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Abstract Multi‐scale calcium (Ca²⁺) dynamics, exhibiting wide‐ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic‐reticulum (ER)‐targeted Ca²⁺indicator, R‐CatchER, which showed superior kinetics in vitro (k_off≥2×10³ s⁻¹,k_on≥7×10⁶ M⁻¹ s⁻¹) and in multiple cell types. R‐CatchER captured spatiotemporal ER Ca²⁺dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca²⁺oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca²⁺‐based functional cooperativity of CaSR. We elucidate the mechanism of R‐CatchER and propose a principle to rationally design genetically encoded Ca²⁺indicators with a single Ca²⁺‐binding site and fast kinetics by tuning rapid fluorescent‐protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G‐CatchER2, an upgrade of our previous (G‐)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca²⁺dynamics, and drug discovery. 

Serine proteases have been proposed to dynamically sample inactive and active conformations, but direct evidence at atomic resolution has remained elusive. Using nuclear magnetic resonance (NMR), we identified a single residue, D164, in exfoliative toxin A (ETA) that acts as a molecular “switch” to regulate global dynamic sampling. Mutations at this site shift the balance between inactive and active states, correlating directly with catalytic activity. Beyond identifying this dynamic switch, we demonstrate how it works in concert with other allosterically coupled sites to rationally control enzyme movements and catalytic function. This study provides a framework for linking conformational dynamics to function and paves the way for engineering enzymes, in particular, proteases, with tailored activities for applications in medicine and biotechnology.