Human cardiac ryanodine receptor: preparation, crystallization and preliminary X-ray ANALysis of the N-terminal region.

ABSTRACT

Human ryanodine receptor 2 (hRyR2) is a calcium ion channel present in the membrane of the sarcoplasmic reticulum of cardiac myocytes that mediates release of calcium ions from the sarcoplasmic reticulum stores during excitation- contraction coupling. Disease-causing mutations of hRyR2 are clustered into N-terminal (amino acids 1-600), central (amino acids 2100-2500) and C-terminal (amino acids 3900-5000) regions. These regions are believed to be involved in regulation of channel gating. The N-terminal region of hRyR2 has been implicated in regulating basal channel activity by interaction with the central hRyR2 region. This paper reports preparation, crystallization and preliminary X-ray analysis of recombinant hRyR2(1-606) N-terminal fragment. Soluble hRyR2(1-606) was expressed in Escherichia coli. Purification conditions were optimized using thermal shift assay. The quality and stability of the sample was probed by dynamic light scattering. A monomeric protein showing over 95% purity was obtained. The protein was crystallized by the hanging drop vapor-diffusion method. Diffraction data with resolution 2.39 Å were collected and processed.