Wrocław University of Science and Technology, Faculty of Chemistry, Department of Biochemistry, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland.
A. Mickiewicz University, Faculty of Physics, Molecular Biophysics Division, Umultowska 85, 61-614, Poznań, Poland.
Biochim Biophys Acta Proteins Proteom. 2017 Nov;1865(11 Pt A):1358-1371. doi: 10.1016/j.bbapap.2017.08.019. Epub 2017 Sep 1.
Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.
鱼耳石是参与听觉和平衡感的碳酸钙生物矿物质。有机基质在其形成中起着至关重要的作用。耳石基质大分子-64（OMM-64）是虹鳟鱼耳石中发现的一种高度酸性、钙结合蛋白（CBP）。它是高分子量聚集体的一个组成部分，影响体外碳酸钙的大小、形状和多态性。在这项研究中，开发了一种有效表达和纯化 OMM-64 的方案。首次描述了 OMM-64 的完整结构特征。结合使用各种生物物理方法表明，OMM-64 以固有无序单体的形式存在。在变性条件（pH、温度）下，OMM-64 表现出折叠倾向。确定 OMM-64 以毫摩尔亲和力结合约 61 个钙离子。折叠-去折叠实验表明，钙离子诱导 OMM-64 的崩溃。研究了鱼内淋巴中存在的其他抗衡离子对 OMM-64 构象变化的影响。讨论了 OMM-64 无规性质的意义和该蛋白的可能功能。
