Aitio H, Annila A, Heikkinen S, Thulin E, Drakenberg T, Kilpeläinen I
Institute of Biotechnology/NMR Laboratory, University of Helsinki, Finland.
Protein Sci. 1999 Dec;8(12):2580-8. doi: 10.1110/ps.8.12.2580.
Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.
芹菜红素是一种从革兰氏阳性细菌糖多孢红霉菌中分离出的20 kDa钙结合蛋白。基于氨基酸序列同源性，有人提出芹菜红素属于无脊椎动物肌浆EF-手型钙结合蛋白（SCPs）家族，因此预计它具有钙缓冲功能。核磁共振光谱用于获取该蛋白在溶液中的结构信息。通过三共振实验HNCACB、HN(CO)CACB、HNCO、CC(CO)NH和[15N]编辑的TOCSY以及HCCH-TOCSY获得了主链和侧链的1H、13C和15N归属。利用二级化学位移和特征性核Overhauser效应（NOE）确定二级结构。此外，从溶解在稀液晶中的样品获得的自旋态选择性[1H, 15N]相关光谱测量了主链N-H剩余偶极耦合。观察到四个具有特征性螺旋-环-螺旋模式的EF-手基序。其中三个是典型的钙结合EF-手，而位点2是一个非典型的非结合位点。通过偶极耦合评估了芹菜红素的整体折叠。将测得的偶极耦合与从与芹菜红素序列同源的四种蛋白质晶体结构计算得到的值进行比较。这些数据使我们能够识别芹菜红素与多毛纲动物杂色沙蚕和文昌鱼文昌鱼的肌浆钙结合蛋白折叠之间的总体相似性。
