Green B N, Kuchumov A R, Walz D A, Moens L, Vinogradov S N
Micromass Ltd., Tudor Road, Altrincham, Cheshire WA14 5RZ, U.K.
Biochemistry. 1998 May 5;37(18):6598-605. doi: 10.1021/bi972644y.
The quaternary structure of the cysteine-rich, approximately 3500-kDa chlorocruorin (Chl) from the marine polychaete Eudistylia vancouverii was investigated using maximum entropy deconvolution of the electrospray ionization mass spectra (ESIMS). The native Chl provided two groups of peaks, at approximately 25 and approximately 33 kDa, and one peak at approximately 66 kDa. ESIMS of the reduced and reduced and carbamidomethylated Chl and of its subunits obtained by HPLC provided the complete subunit composition of the Chl. Two groups of nonglobin linker chains were observed: L1a-f (25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25 096.8 Da) and L2a-d (25 402.7, 25 446.0, 25 461.6 and 25 478.3Da) (+/-2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains were found, a1, a2, and b1-4, with reduced masses of 16 051.5, 16 172.4, 16 853.5, 17 088.9, 17 161.2 and 17 103.6 (+/-1.0 Da) and relative intensities of 8:4:1:4:2:1, respectively. Disulfide-bonded dimers and a tetramer of globin chains were identified: D1 = a1 + b3 at 33 207.1; D2 at 33 374.1, which had a cysteinylated Cys (a2 + b2 + Cys); and D3 = a1 + b4 at 33 149.4 Da (+/-3.0 Da), with relative intensities D1:D2:D3 = 5:4:1 and T = a1 + a2 + b1 + b2 at 66 154.8 +/- 4.0 Da. A 206-kDa dodecamer subunit obtained by dissociation of the Chl in 4 M urea [Qabar, A. N., et al. (1991) J. Mol. Biol. 222, 1109-1129], was found to consist only of tetramers T. A model was proposed for the Chl, based on a dimer:tetramer ratio of 2:1: four 206-kDa dodecamers (trimer of tetramers) and 48 dimers tethered to a framework of 30 L1 and 12 L2 linker chains. The 144 globin chains (2480 kDa) and 42 linker chains (1059 kDa) provide a total mass of 3539 kDa, in good agreement with the 3480 +/- 225 kDa determined previously by STEM mass mapping. The hierarchy of disulfide-bonded globin subunits observed for Eudistylia Chl provides a built-in heterogeneity of hexagonal bilayer structures.
利用电喷雾电离质谱（ESIMS）的最大熵去卷积技术，对来自海洋多毛纲动物温哥华真蛰虫的富含半胱氨酸、分子量约为3500 kDa的血绿蛋白（Chl）的四级结构进行了研究。天然Chl产生两组峰，分别位于约25 kDa和约33 kDa处，以及一个位于约66 kDa处的峰。还原型、还原并氨甲酰甲基化型Chl及其通过高效液相色谱法获得的亚基的ESIMS给出了Chl完整的亚基组成。观察到两组非球蛋白连接链：L1a - f（25000.4、25017.9、25039.6、25057.0、25074.以及25096.8 Da）和L2a - d（25402.7、25446.0、25461.6和25478.3 Da）（±2.5 Da），相对强度L1:L2 = 5:2。发现了六条球蛋白链，a1、a2和b1 - 4，还原后的质量分别为16051.5、16172.4、16853.5、17088.9、17161.2和17103.6（±1.0 Da），相对强度分别为8:4:1:4:2:1。鉴定出了二硫键连接的球蛋白链二聚体和四聚体：D1 = a1 + b3，质量为33207.1；D2质量为33374.1，其含有一个半胱氨酸化的半胱氨酸（a2 + b2 + Cys）；D3 = a1 + b4，质量为33149.4 Da（±3.0 Da），相对强度D1:D2:D3 = 5:4:1，T = a1 + a2 + b1 + b2，质量为66154.8 ± 4.0 Da。通过在4 M尿素中解离Chl得到的一个206 kDa的十二聚体亚基[Qabar, A. N., 等人（1991年）《分子生物学杂志》222卷，1109 - 1129页]，被发现仅由四聚体T组成。基于二聚体:四聚体比例为2:1，提出了一个Chl的模型：四个206 kDa的十二聚体（四聚体的三聚体）和48个二聚体连接到由30条L1和12条L2连接链组成的框架上。144条球蛋白链（2480 kDa）和42条连接链（1059 kDa）的总质量为3539 kDa，与先前通过扫描透射电子显微镜（STEM）质量图谱测定的3480 ± 225 kDa高度吻合。在温哥华真蛰虫Chl中观察到的二硫键连接的球蛋白亚基的层级结构提供了六方双层结构的内在异质性。
Zotero 插件