短链蝎毒素BmK38的1^H—NMR谱峰归属、二级结构分析及溶液构象

Sequence-specific assignments of proton NMR resonance peaks,analysis of secondary structural elements and solution conformation of BmK38

BmK38是最近从东亚钳蝎（Buthus martensi Karsch，BmK）中分离纯化得到的一种短链蝎毒素．应用2D-NMR技术研究BmK38的溶液构象，通过分析其在水溶液中的DQF-COSY、TOCSY和NOESY等1^H—NMR谱，识别了BmK38全部40个氨基酸残基的自旋体系，并通过分析NOESY谱图中dαN、dNN、dβN的联系，完成了序列专一性谱峰归属，标定了全部主链质子和绝大部分侧链质子的化学位移．根据谱峰归属的结果和NMR数据分析了BmK38的二级结构组成并且计算出BmK38的溶液构象．结构计算的结果与前述二级结构的分析是一致的．结果表明，肽段Gln8-Arg17形成α螺旋，而肽段Gly22-Glu29以及Leu32-CYS39构成反平行的口折叠，属于典型的短链蝎毒素的折叠形式．通过与其他OUKTx短链蝎毒素的结构比较，讨论了BmK38的结构特异性以及结构功能的相互关系．

BmK38 is a novel 40-amino acid residue peptide identified from the venom of Chinese scorpion Buthus martensi Karsch. The primary sequence analysis of BmK38 shows that it is a cysteine-rich peptide, and the number of residues between the fourth and the fifth cysteine residue is much bigger than that of other a-KTx toxins. Its theoretical MW and theoretical PI are 4 558 and 8. 64, respectively. BrnK38 was synthesized on Boc-Lys （2CIZ）-OCH2-PAM resin using a custom-modified, machine-assisted chemistry tailored from the published in situ DIEA neutralization/HBTU activation protocol for Boc solid phase peptide synthesis and then purified by reversed-phase HPLC to homogeneity. In order to investigate the structure-function relationship, two dimensional homonuclear 1^H NMR technique was used to determine the structure of BmK38, By means of two-dimensional DQF-COSY, TOCSY and NOESY spectroscopies, spin systems of all 40 residues were identified. Then the sequence-specific assignment was completed by analyzing the dαN ,dNN and dβN connectivities. The chemical shifts of all the backbone protons and most of the side-chain protons were subsequently identified. Both the dαN, dNN and dβN connectivities and the chemical shift index （CSI） data show that Bmk38 forms a typical α/β scaffold adopted by most short-chain scorpion toxins. At last, the solution structure was determined by using the standard simulated annealing and energy minimization protocols. The results show that the secondary structure of BmK38 consists of a two-stranded antiparallel β-sheet from residues 22-39, and an α-helix from residues 8- 17. The structure comparison of BmK38 with other α-KTx toxins, especially the unique β-turn, may provide an explanation of the pilot study of its physiological function.