XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Posters
73.
THE STUDY of rYaNODINE rECEPTOr 2 N-TErMINal rEGION
rESPONSIBLE for HEart arrYTHMIas aND HEart faILUre
Ľubomír Borko 1,2 , Vladena Bauerová-Hlinková 2 , Eva Hostinová 2 ,
Juraj Gašperík 2 and Jozef Ševčík 2
1
Department of Molecular Biology, PRIF UK, Mlynská dolina, 842 15 Bratislava
2
Institute of Molecular Biology, SAV, Dúbravská cesta 21, 845 51 Bratislava
Ryanodine receptor (RyR) is a homotetramer composed of four subunits with a molecular
weight of ~560 kDa. In humans there are three isoforms of RyR: RyR1, RyR2 and RyR3.
RyR1 (expressed mostly in sceletal muscle) and RyR2 (in myocardium) are suggested to
be of a vital importance. RyRs are localized in the membrane of sarcoplasmatic reticulum.
The role of RyR is to transport Ca 2+ from sarcopasmatic reticulum to the myoplasm.
The main RyR2 gating regulation mechanism is considered to be the domain-domain
interaction between the N – terminal (aa ~ 1-600) and central region (aa ~ 2100-2500).
A hypothesis was proposed that mutations in these regions cause regulation failure and
thus lead to nonspecific Ca 2+ release which results in several heart diseases. Ryanodine
receptor 2, has a domain structure, so we decided to prepare DNA fragments coding
for residues 384-606, 391-606, 409-606, 1-606 and 1-655. These fragments were cloned
into appropriate pET expression vectors for heterologous expression in E. coli strains.
Recombinant polypeptides RyR2 409-606, NusA_409-606, Trx_409-606, Trx_391-606,
Trx_384-606, 1-606 and 1-655 were obtained in high expression levels. Because of high
rate of aggregation and instability of these recombinant proteins it was necessary to
use appropriate detergents, pH values, buffers and salts. Isolation and purification of
recombinant polypeptides was optimized to obtain soluble monomeric products. To
study stability and folding we prepared a number of N-terminal mutations (1-606 fragment),
which are connected to heart arrhythmias and failures. Our work is oriented to
study domain-domain and domain-ligand interactions and structure determination of
interacting regions by X-ray crystallography.
196 XXII. Biochemistry Congress, Martin