Human Cardiac Ryanodine Receptor: Preparation, Crystallization and Preliminary X-ray Analysis of the N-terminal Region

Title:Human Cardiac Ryanodine Receptor: Preparation, Crystallization and Preliminary X-ray Analysis of the N-terminal Region

Volume: 20 Issue: 11

Author(s): L’ubomír Borko, Július Kostan, Alexandra Zahradníkova, Vladimír Pevala, Juraj Gasperík, Eva Hostinova, L’ubica Urbanikova, Kristina Djinovic-Carugo, Vladena Bauerova-Hlinkova and Jozef Sevcík

Affiliation:

Keywords: Crystallization, dynamic light scattering, human cardiac ryanodine receptor (hRyR2), protein purification, thermal shift assay.

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.

Cite this article as:

Borko L’ubomír, Kostan Július, Zahradníkova Alexandra, Pevala Vladimír, Gasperík Juraj, Hostinova Eva, Urbanikova L’ubica, Djinovic-Carugo Kristina, Bauerova-Hlinkova Vladena and Sevcík Jozef, Human Cardiac Ryanodine Receptor: Preparation, Crystallization and Preliminary X-ray Analysis of the N-terminal Region, Protein & Peptide Letters 2013; 20 (11) . https://dx.doi.org/10.2174/0929866511320110004