Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii.
Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii.
复制标题
珠蛋白复合物的层次结构。
DOI：
10.1016/0022-2836(91)90596-x
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发表时间：
1991
影响因子：
5.6
通讯作者：
Vinogradov,SN
中科院分区：
文献类型：
--
作者：
Qabar,AN;Stern,MS;Walz,DA;Chiu,JT;Timkovich,R;Wall,JS;Kapp,OH;Vinogradov,SN
通过扫描透射电子显微镜（STEM）对阴性染色标本进行测定，发现多毛类Eudistylia vagiverii细胞外六角双层氯红蛋白的分子尺寸为直径27.5 nm，高度18.5 nm。未染色的冻干标本的STEM质量测量提供了3480 ± 225 kDa的分子量（Mm）。叶绿素不含碳水化合物，铁含量为0.251 ± 0.021wt%，最小分子量为22.4kDa。通过质谱和核磁共振谱的分析，确定了该化合物为3位带有甲酰基的血红素IX。SDS/聚丙烯酰胺凝胶电泳、反相层析和N-末端测序结果表明，该蛋白至少由3条~30 kDa的亚基链和5条~16 kDa的亚基链组成，两种亚基的比例为0·26：0·74（± 0·08）。在尿素或盐酸胍存在下，在中性pH值下氯红蛋白完全解离，然后进行凝胶过滤，产生由三个峰（B、C和D）组成的洗脱谱。级分B和C由~16 kDa链组成，级分D由~30 kDa亚基组成。未染色的冻干级分B和C的STEM图像中颗粒的质量测量分别提供208 ± 23 kDa和65 ± 12 kDa的Mm，与通过凝胶过滤获得的191 ± 13 kDa和67 ± 5 kDa一致。Mm = 221 + 21 kDa的颗粒也观察到未染色的，冷冻干燥的chlorocruorin的STEM图像。这些结果意味着，除了具有0.26至0.47血红素基团/链的~30 kDa接头亚基之外，氯红蛋白结构还包括珠蛋白链的~65 kDa四聚体和~200 kDa十二聚体（四聚体的三聚体）。从摩尔氨基酸组成计算的四聚体和接头亚基的化学计量为34 ± 4和43 ± 9。氯红蛋白的完全解离伴随着55 ± 14 Ca 2 +/mol蛋白质的50 - 75%损失，并且通过10 - 25 mm-Ca 2+的存在降低至~ 35%。在2·5 ~ 5 mm-Ca ~（2+）存在下，解离的氯红蛋白的再结合达到最大。在不存在或存在Ca 2+的情况下，十二聚体和/或四聚体亚基仅在接头亚基存在下表现出非常有限的（<10%）重新缔合成六边形双层结构。虽然氯红蛋白和十二聚体亚基的圆二色性（200 - 250 nm）相似，α-螺旋含量约为50%，但四聚体和连接亚基的[θ] 222较低。我们的结果与Eudisylachlorocruorin的四级结构是一个球蛋白复合物的等级的观点是一致的，其中整个分子的66 - 75%由12 ~ 200 kDa的十二聚体组成，每个十二聚体由具有不完整的“肌红蛋白折叠”的球蛋白链的四聚体的三聚体组成。十二聚体通过30至40个Ca 2+和30至40个血红素缺陷嵌合珠蛋白链连接成六边形双层结构。
The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaeteEudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27·5 nm and height of 18·5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 ± 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0·251 ± 0·021 wt %, corresponding to a minimumMmof 22·4 kDa. Mass spectra and1nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of ~30 kDa and five chains of ~16 kDa; the two types of subunits occur in the ratio 0·26:0·74(± 0·08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the ~16 kDa chains and fraction D consisted of the ~30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C providedMmof 208 ± 23 kDa and 65 ± 12 kDa, respectively, in agreement with 191 ± 13 kDa and 67 ± 5 kDa obtained by gel filtration. Particles withMm= 221 + 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the ~30 kDa linker subunits that have 0·26 to 0·47 heme groups/chain, comprises ~65 kDa tetramers and ~200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 ± 4 and 43 ± 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 ± 14 Ca2+/mol protein, and was decreased to ~35 % by the presence of 10 to 25 mm-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2·5 to 5 mm-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+exhibited very limited (<10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit. Although the circular dichroism (200 to 250 nm) of the chlorocruorin and the dodecamer subunit was similar, with α-helical contents of ~50%, the [θ]222of the tetramer and linker subunits were lower. Our results are compatible with a view of the quaternary structure ofEudistyliachlorocruorin as a hierarchy of globin complexes, wherein 66 to 75 % of the whole molecule is composed of 12 ~ 200 kDa dodecamers, each consisting of a trimer of tetramers of globin chains having an incomplete “myoglobin fold”. The dodecamers are linked into a hexagonal bilayer structure by 30 to 40 Ca2+and 30 to 40 heme-deficient, chimeric globin chains.