Blayney Lynda, Beck Konrad, MacDonald Ewan, D'Cruz Leon, Nomikos Michail, Griffiths Julia, Thanassoulas Angelos, Nounesis George, Lai F Anthony
Institute of Molecular and Experimental Medicine, Cardiff University, Cardiff, UK.
Biochim Biophys Acta. 2013 Oct;1830(10):4426-32. doi: 10.1016/j.bbagen.2013.05.038. Epub 2013 Jun 6.
This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6).
Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation.
The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found.
The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP.
Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding.
本研究旨在确定心肌兰尼碱受体（RyR2）中央结构域（一个与儿茶酚胺多形性室性心动过速（CPVT）突变相关的区域）是否与RyR2调节剂、ATP和FK506结合蛋白12.6（FKBP12.6）相互作用。
野生型（WT）RyR2中央结构域构建体（G(2236)至G(2491)）以及含有CPVT突变P2328S和N2386I的构建体被表达为重组蛋白。通过圆二色性（CD）光谱和盐酸胍化学变性来检测蛋白质的折叠和稳定性。
远紫外CD光谱显示可溶性稳定折叠蛋白，野生型和突变型蛋白具有相似的二级结构。化学变性分析也证实野生型和突变型构建体的蛋白均稳定，具有相似的两态解折叠。通过荧光光谱法测量ATP和咖啡因结合。ATP和咖啡因的结合EC50约为200 - 400μM，野生型和突变型构建体的亲和力相同。与其他ATP结合蛋白的序列比对表明RyR2中央结构域包含ATP结合口袋的特征。通过戊二醛交联测试中央结构域与FKBP12.6的相互作用，未发现关联。
以“正确”折叠的重组蛋白形式表达的RyR2中央结构域，根据保守ATP结合序列基序的生物信息学证据与ATP结合。与FKBP12.6的相互作用不明显。CPVT突变不会破坏二级结构也不会破坏与ATP的结合。
RyR2中央结构域CPVT突变簇的一部分可以独立表达并保留ATP结合能力。
