FRET-Based Mapping of Three Structural Domains within the Full-Length Ryanodine Receptor Type 1

Abstract

The type 1 ryanodine receptor (RyR1) mediates excitation contraction coupling in skeletal muscle by releasing intracellular calcium in response to cell depolarization. Understanding the structure and conformational dynamics of RyR1 is challenging due to its size (homotetrameric subunit Mr∼560 kDa) and its association with numerous regulatory proteins. However, recent X-ray crystallographic studies indicate that a fragment comprised of the first 200 amino acids of RyR1 folds into a compact β-trefoil domain. Based on structural homology with the inositol trisphosphate receptor, the next 400 amino acids of RyR1 are predicted to fold into a second β-trefoil and an alpha helical domain. In this report, the relative orientation of these three domains (termed here as β1, β2 and α1) 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 β1, β2 and α1 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).