Characterization of N-terminal part of human ryanodine receptor 2

 

V. Bauerová-Hlinková1, E. Hostinová1, J. Gašperík1, J. Bauer1, K. Beck3,

A. Zahradníková1,4 and J. Ševčík1

 

1Institute of Molecular Biology, SAV, Dúbravská cesta 21, 845 51 Bratislava

2Department of Molecular Biology, PRIF UK, Mlynská dolina,  842 15 Bratislava
3Cardiff University School of Dentistry, Cardiff CF14 4XY, UK.
4Institute of Molecular Physiology and Genetics SAV, Vlárska cesta 5, 833 34 Bratislava

 

The ryanodine receptor (RyR) is the calcium release channel of a vital importance, responsible for muscular contraction  in mammalian cells. It is localized on the membrane of the sarcoplasmatic reticulum (SR), where it transfers Ca2+ from SR  into cytoplasm. This channel is composed of a larger cytoplasmic and a smaller transmembrane part and is built up from four polypeptide chains to give a homotetramer with a molecular weight of ~ 500 MDa. Due to its size and properties, the structure of the whole receptor has been determined only by cryoelectron microscopy [1,2]. An X-ray crystal structure has been determined for only the first 217 aa. [3] and for the first 532 residues of RyR1 [4].

In this work we have focused predominantly on the domain analysis of the N-terminal region (residues 1–759) of the human cardiac ryanodine receptor (RyR2) which includes RyR2 mutation cluster associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). In our strategy we used a bioinformatics approach followed by protein expression, solubility analysis and limited proteolytic digestion [5,6]. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for several N-terminal RyR2 fragments. The purity, monomeric state, folding and stability of these proteins were verified by SDS PAGE, gel filtration, dynamic light scattering measurements, Circular dichroism and thermofluor shift assay. Moreover, based on the similarity between RyR1 and RyR2 isoforms, we performed a homology modeling of the N-terminal fragment of RyR2 (aa. 1 – 553). All these approaches help us to understand better structure-function relationship of this protein.

Acknowledgement. This work was supported by the research grants from the Slovak Grant Agency VEGA No. 2/0131/10 and APVV-0628-10.

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