Troponin I encompasses an extended troponin C in the Ca(2+)-bound complex: a small-angle X-ray and neutron scattering study.
Troponin I encompasses an extended troponin C in the Ca(2+)-bound complex: a small-angle X-ray and neutron scattering study.
复制标题
肌钙蛋白 I 在 Ca(2 ) 结合复合物中包含延伸的肌钙蛋白 C：小角度 X 射线和中子散射研究。
DOI：
10.1021/bi00193a009
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Trewhella,J
中科院分区：
文献类型：
--
作者：
Olah,GA;Rokop,SE;Wang,CL;Blechner,SL;Trewhella,J
材料和方法样品制备.中子散射实验涉及测量“对比系列”，其中氘代4Ca 2 +-TnC和非氘代TnI的复合物溶解在具有不同D2 Q：H2 Q比率的溶剂中。选择TnC组分的部分氘化，以使整个络合物的溶剂匹配点在65和72%D20之间，从而允许在溶剂匹配点的任一侧进行中子散射测量。使用从S.希区柯克-德格雷戈里（新泽西医学和牙科大学，罗伯特伍德约翰逊医学院）。TnC基因在大肠杆菌BL 21（DE 53）plysS中的pET 3d载体中。使用M9盐和含有氘化和非氘化成分的有机补充剂，使细胞在75-80% D20中生长。氘代有机组分来自Merck氘代全藻水解物，Merck报道为60-65%氨基酸、10-15%糖和30-35%盐。通过氨基酸分析确定了水解产物的氨基酸组成。用非氘代氨基酸和葡萄糖稀释水解产物，得到3：1比率的氘代与非氘代氨基酸和糖。最终有机添加量为10 g/L。培养基还含有20 mg/L氨苄青霉素和25 mg/L氯霉素。细胞在BioFlo II发酵罐（新玩法科技公司）中于37 ℃生长至Am为0.4.通过添加IPTG至0.4mM的浓度来诱导TnC表达。在Am下监测生长直至其稳定，通常在3-4小时后约Am= 2.4。通过在Beckman J21 C中用J-14转子以12000 rpm离心15分钟收获细胞，并将沉淀物在-80 ℃冷冻过夜。根据Xu和Hitchcock-Degregori（1988）的方法进行几处修改来纯化TnC。他们描述的硫酸铵分馏总是进行，然后是苯基-琼脂糖柱和HPLC分离步骤。合并苯基-琼脂糖洗脱份，用0.001 M NH 4 HCO 3透析以除去盐，并将透析液冻干。将冻干粉末重悬于20 mM磷酸钠缓冲液（pH 6.5）、0.1 mM CaCl 2中，并应用于Pharmacia ProRPC HR 5/10反相HPLC柱。列后
MATERIALS AND METHODSSample Preparation. The neutron scattering experiment involved measuring a “contrast series” in which a complex of deuterated 4Ca2+-TnC and nondeuterated Tnl is solubilized in solvents with differentD2Q: H2Q ratios. Partial deuteration of the TnC component was chosen in order to have the solvent match point for the overall complex between 65 and 72% D20, thus allowing neutron scattering measurements on either side of the solvent match point. Partially deuterated TnC was produced using a bacterial expression system obtained from S. Hitchcock-DeGregori (University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School). The TnC gene is in a pET 3d vector in Escherichia coli BL21 (DE53) plysS. The cells were grown in 75-80% D20 using M9 salts with an organic supplement containing deuterated and nondeuterated com-ponents. The deuteratedorganic components came from Merck deuterated whole algal hydrolysate, reported by Merck to be 60-65% amino acids, 10-15% sugars, and 30—35% salts. The amino acid composition of the hydrolysate was determined by amino acid analysis. The hydrolysate was diluted with nondeuterated amino acids and glucose to give a 3: 1 ratio of deuterated to nondeuterated amino acids and sugars. The final organicsupplement was 10 g/L. The media also contained 20 mg/L ampicillin and 25 mg/L chloramphenicol. Cells were grown at 37 C to an Am of 0.4 in a BioFlo II fermentor (New Brunswick Scientific). TnC expression was induced by addition of IPTG to a concentration of 0.4 mM. Growth was monitored at Am until it leveled off, usually at about Am= 2.4 after 3-4 h. Cells were harvested by centrifugation in a Beckman J21C with a J-14 rotor for 15 min at 12 000 rpm, and the pellet was frozen overnight at-80 C.TnC was purified according themethod of Xu and Hitchcock-Degregori (1988) with several modifications. The ammonium sulfate fractionation they describe was always carried out, followed by the phenyl-Sepharose column and HPLC separation steps. The phenyl-Sepharose fractions were combined and dialyzed against 0.001 M NH4HCO3 to remove salts, and the dialysate was lyophilized. The lyophilized powder was resuspended in 20 mM sodium phosphate buffer, pH 6.5, 0.1 mM CaCl2, and applied to a Pharmacia ProRPC HR 5/10 reverse-phase HPLC column. After the column