Folding and binding kinetics of calnuc: insights into its potential as a first responder to calcium signals and conversion into a novel transducing element for ultrafast calcium biosensors
Abstract
Calnuc (nucleobindin-1) is a ubiquitously expressed Ca2+ binding protein withnumerous binding partners and functions throughout the cell. Some of these functions are regulated by Ca2+ binding to calnuc, which is known to induce folding. However, these Ca2+ dependent processes occur in areas of the cell with a 3000 fold difference in concentration which raises questions about how calnuc can regulate its binding partners across such a broad range. It is also unclear how its conformational dynamics relate to these functions at different Ca2+ concentrations.
We use confocal single molecule FRET experiments to study the binding andconformational kinetics of calnuc’s Ca2+ binding domain. This technique allows us to resolve populations of each conformational state and their distributions across the physiological Ca2+ range. We combine this with a maximum likelihood analysis to build a model for elucidating the interplay between calnuc’s binding and conformational dynamics. The modeled behavior provides new insights into the populations of each state in different Ca2+ signaling scenarios and how they relate to calnuc’s functions, including regulation of G-proteins. This analysis also reveals conformational kinetics for calnuc on the order of milliseconds. When paired with Ca2+-induced folding, calnuc is an ideal transducer for ultra-fast Ca2+ biosensing with faster rates that existing genetically encodable Ca2+ indicators (GECIs).
We create the first ever calnuc-based GECI and apply consensus protein designto generate improved variants. Together, these can detect Ca2+ signals for the entire physiological range and include variants with high affinity, medium affinity, or an ultrabroadband sensing range. These fast molecular scale biosensors are encoded into HEK293FT and Jurkat cells to measure their in vivo responses to increased Ca2+. We develop a method for continuous measuring of a cell population’s response to stimuli using flow cytometry and use it to measure Ca2+ signals in Jurkat cells activated via their T cell receptor, demonstrating the capability of our biosensors.