A FRET-based Assay To Measure Molecular Distances Within The Ryanodine Receptor Type 1 (RyR1)

Abstract

The type 1 ryanodine receptor (RyR1) is an intracellular Ca2+-release channel that mediates excitation contraction coupling in skeletal muscle. This enormous homotetrameric protein has a subunit molecular weight of 565 kDa and is associated with numerous regulatory proteins that modulate its function in vivo. Understanding the structure and conformational dynamics of this immense macromolecular complex is a key topic in skeletal muscle biology. In this report, a novel structural assay has been devised that relies upon Forster resonance energy transfer (FRET) to measure distances between defined primary sequence elements of RyR1. These FRET measurements require site-specific incorporation of a fluorescence donor and a fluorescence acceptor into the primary sequence of RyR1. In this system, green fluorescent protein (GFP) fused to the N-terminus of RyR1 acts as the fluorescence donor. The FRET acceptor is an affinity reagent that site-specifically binds to poly-histidine segments (i.e. His tags) engineered into RyR1 where it can accept fluorescence energy from the N-terminal GFP. This report describes the characterization of the FRET acceptor as well as the recombinant His-tagged GFP-RyR1 fusion proteins used for these measurements. In addition, experiments measuring FRET from the N-terminal GFP to the FRET acceptor targeted to His tags introduced into 3 primary sequence elements poorly conserved among the 3 RyR isoforms (i.e. divergent regions) are described. (Supported by NIH grants K01ARO52120 and R21ARO56406).