Conformational variation of calcium-bound troponin C.
Conformational variation of calcium-bound troponin C.
复制标题
钙结合肌钙蛋白的构象变化 C.
DOI：
10.1002/(sici)1097-0134(19991201)37:4
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
PhillipsJr,GN
中科院分区：
文献类型：
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作者：
Soman,J;Tao,T;PhillipsJr,GN
材料和方法。如前所述表达和纯化此处研究的突变Cys 98 Leu兔骨骼肌TnC。7通过悬滴法，在4 ℃下从含有50- 60%MPD、50 mM乙酸钠pH 5.6和5 mM CaCl 2的溶液中通过微接种生长晶体。晶体的空间群为P21，a = 32.1，B = 59.4，c = 47.8 π，c = 101.1，每个不对称单元对应一个分子，马修斯系数为2.4 π 3/Da。8初始电子密度图显示了双叶结构，Se位置的簇可以与apo TnC结构中的两个结构域相关联。使用鸡TnC的Ca 2-饱和平均NMR结构作为起始模型，使用XPLOR 9通过几轮位置和约束温度因子细化来细化结构。2FoFc图谱显示所有四种Ca 2离子的清晰密度。最终结果对应于156个残基和162个水分子的R 25.5%和Rfree 36.0%。坐标和结构因子存放在蛋白质数据库中。（分别为R1 TCF和R1 TCFSF。）结果和讨论。正如其他工作者所示，4-Ca 2兔TnC的整体结构与2-Ca 2禽TnC相似，2-Ca 2禽TnC是一种延长螺旋蛋白，由两个结构域组成，由一个九转螺旋连接。该结构中总共有九个螺旋。N-结构域包含N（1-10）; A（13-26）; B（36-46）; C（52-62）;和D（72-83）螺旋。C结构域包含E（93-102）; F（112-122）; G（128-138）;和H（148-156）螺旋。每个结构域在两个螺旋-环-螺旋钙结合基序中填充有两个Ca 2离子。在4-Ca 2 TnC相对于apo TnC的Δ距离图中存在两个主要特征。首先，螺旋B和接头远离螺旋N/A有大的移动。第二，残基35-57的延伸远离70-83。这对应于螺旋的B/C对和它们之间的接头远离D-螺旋移动。因此，从N-结构域的apo状态到Ca 2-饱和状态的转变的显著特征是螺旋B/C相对于A/D的大的相对运动，导致其采用开放构象。没有涉及C结构域的突出特征，表明它们在两种结构中的构象基本相同。
Materials and Methods. The mutant Cys98Leu rabbit skeletal TnC studied here was expressed and purified as previously described. 7 Crystals were grown by the hanging-drop method by microseeding at 4 C from solutions containing 50–60% MPD, 50 mM sodium acetate pH 5.6 and 5 mM CaCl2. The crystals were characterized as belonging to the space group P21 with a 32.1, b 59.4, c 47.8 Å, 101.1 and one molecule per asymmetric unit based on a Matthews’ coefficient of 2.4 Å3/Da. The structure was determined using anomalous selenomethionine data and MADSYS. 8 The initial electron density maps showed a bilobed structure, and the clusters of Se positions could be correlated with the two domains in the apo TnC structure. Using the Ca2-saturated average NMR structure of chicken TnC as the starting model, the structure was refined using XPLOR9 with several rounds of positional and restrained temperature factor refinements. 2FoFc maps showed clear density for all four Ca2 ions. The final results correspond to R 25.5% and Rfree 36.0% for 156 residues and 162 water molecules. Coordinates and structure factors were deposited at the Protein Data Bank.(Entries 1TCF and R1TCFSF respectively.)Results and Discussion. As shown by other workers, the overall structure of 4-Ca2 rabbit TnC is similar to that seen in 2-Ca2 avian TnC, an elongated-helical protein consisting of two domains connected by a nine-turn helix. There are a total of nine helices in the structure. The N-domain contains the N (1–10); A (13–26); B (36–46); C (52–62); and D (72–83) helices. The C-domain contains the E (93–102); F (112–122); G (128–138); and H (148–156) helices. Each domain is filled with two Ca2 ions in the two helix-loop-helix calcium-binding motifs. There are two dominant features in a delta-distance-plot of 4-Ca2 TnC vs. apo TnC. First, there is a large movement of helix B and the linker away from helices N/A. Second, the stretch of residues 35–57 moves away from 70–83. This corresponds to the B/C pair of helices and the linker between them moving away from the D-helix. Therefore, a significant feature of the transition from the apo state of the N-domain to the Ca2-saturated state is the large, relative movement of helices B/C with respect to A/D, causing it to adopt an open conformation. There are no prominent features involving just the C-domains, suggesting that their conformations in the two structures are essentially identical.