Localization of the Dantrolene Target Sequence in the Cardiac Ryanodine Receptor by Combining cryo-EM and FRET Analysis

Abstract

amino acids of RyR1 are predicted to fold into a second b-trefoil and an alpha helical domain. In this report, the relative orientation of these three domains (termed here as b1, b2 and a1) within the full-length RyR1 protein has been investigated using a novel FRET-based technique. This method monitors the relative proximity of a GFP fluorescent donor fused into RyR1 and a fluorescent acceptor, Cy3NTA, targeted to poly-histidine (His) tags inserted into the primary sequence of RyR1. In this study, GFP was fused to single positions within b1, b2 and a1 and FRET was then measured to Cy3NTA targeted to 6 His tags singly introduced into these three domains. The results of these measurements provide a detailed picture of the relative orientation of these three sub-domains within full-length RyR1 expressed in a cellular context. Further structural studies using this technique can now be undertaken to determine how these domains move during channel gating. (Supported by NIH grant R21ARO56406). 1030-Plat3D Mapping of the SPRY2 Domain of RyR1 by Antibody Labeling and Single-Particle cryo-EM