Proximity mapping the surface of a membrane protein using an artificial protease: demonstration that the quinone-binding domain of subunit I is near the N-terminal region of subunit II of cytochrome bd.
Proximity mapping the surface of a membrane protein using an artificial protease: demonstration that the quinone-binding domain of subunit I is near the N-terminal region of subunit II of cytochrome bd.
复制标题
使用人工蛋白酶绘制膜蛋白表面的邻近图谱：证明亚基 I 的醌结合域位于细胞色素 bd 亚基 II 的 N 末端区域附近。
DOI：
10.1021/bi00036a002
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Gennis,RB
中科院分区：
文献类型：
--
作者：
Ghaim,JB;Greiner,DP;Meares,CF;Gennis,RB
1995年7月24日收到的修订版Mandalpt®摘要：一个新的实验已被用来显示大肠杆菌细胞色素bdquinol氧化酶表面上的位点之间的邻近关系。将人工蛋白酶铁（S）-1-[对-（溴乙酰氨基）苄基]-EDTA（Fe-BABE）缀合至位于亚基I或亚基II中的所选反应性半胱氨酸，目的是鉴定每个连接位点的12%内的氨基酸残基。用H2O2和抗坏血酸活化蛋白酶几秒钟，分离水解产物并通过N-末端测序进行分析。在其他结果中，我们发现，亚基II的残基39是在推定的醌结合结构域（Q环）的氧化酶的亚基I的残基255附近。由于这种技术对氨基酸侧链的性质不敏感，因此在揭示复杂蛋白质亚基内部和亚基之间的空间关系方面，它应该被证明是普遍有价值的，而在这些复杂蛋白质中，高分辨率的结构信息是不可用的。如下所示，通过使用试剂Fe-BABE可以获得特定氨基酸残基邻近度的信息。1将单个反应性半胱氨酸残基置于目标位置后，可以连接Fe-BABE并用于水解附近的肽键。这些键可以位于多亚基复合物的相同亚基内或不同亚基中。它们在蛋白质一级序列中的位置可以通过鉴定水解时形成的新的N-末端来确定。因此，有用的约束有关的三维折叠的子单元和有关的子单元界面区域可以导出。由于通过这种技术鉴定为邻居的残基必须位于膜的同一侧，因此这也提供了对所提出的预测每个多肽的哪些区域位于膜的每一侧的二维模型的实验检查。
Revised Manuscript Received July 24, 1995® abstract: A novel experiment has been used to show proximity relationships between sites on the surface of the cytochrome bdquinol oxidase of Escherichia coli. The artificial protease iron (S)-l-[p-(bromoacetamido) benzyl]-EDTA (Fe—BABE) was conjugated to selected reactive cysteines placed in subunit I or subunit II, with the aim of identifying amino acid residues within% 12 A of each site of attachment. The protease was activated with H2O2 and ascorbate for a few seconds, and hydrolysis products were isolated and analyzed by N-terminal sequencing. Among other results, we found that residue 39 of subunit II is near residue 255 of subunit I in the putative quinone-binding domain (Q loop) of the oxidase. Since this technique is insensitive to the nature of the amino acid side chains, it should prove generally valuable in revealing spatial relationships both within and between subunits in complex proteinswhere high-resolution structural information is not available.Obtaining structural information about membrane proteins is a major experimental challenge. As demonstrated below, information on the proximity of particularamino acid residues can be gainedthrough the use of the reagent Fe—BABE. 1 After a single reactive cysteine residue is placed in a position of interest, Fe-BABE can be attached and used to hydrolyze nearby peptide bonds. These bonds can be located within the same subunit or in a different subunit of a multisubunit complex. Their position in the primary sequence of the protein can be determined by identifying the new N-termini formedupon hydrolysis. Hence, useful constraints concerning the three-dimensional folding of the subunits and about the subunit interface regions can be derived. Since residues that are identified as neighbors by this technique must be located on the same side of the membrane, this also provides an experimental check on proposed two-dimensional models predicting which regions of each polypeptide are on each side of the membrane.
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DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
大肠杆菌末端氧化酶中细胞色素 b-558、b-595 和 d 之间的电子流和血红素-血红素相互作用。
DOI：
10.1016/0005-2728(87)90050-8
发表时间：
1987
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
A. Hata;K. Matsuura;S. Itoh;Y. Anraku
通讯作者：
Y. Anraku
大肠杆菌细胞色素 d 复合物的胰蛋白酶蛋白水解选择性抑制泛醇氧化酶活性，但不影响 N,N,N,N-四甲基对苯二胺氧化酶活性。
DOI：
10.1016/s0021-9258(18)60711-2
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
R. Lorence;K. Carter;R. Gennis;K. Matsushita;H R Kaback
通讯作者：
H R Kaback
影响因子：
3.2
作者：
HILL, S;VIOLLET, S;ANTHONY, C
通讯作者：
ANTHONY, C
芬顿氧化机制：生物相关底物与两种氧化中间体的反应性与羟基自由基的预测不同。
DOI：
10.1073/pnas.91.14.6604
发表时间：
1994
影响因子：
11.1
作者：
David A. Wink;Raymond W. Nims;Joseph E. Saavedra;W. E. Utermahlen;Peterc . Fords
通讯作者：
Peterc . Fords