Electrochemical identification of dopachrome isomerase in Drosophila melanogaster.
Electrochemical identification of dopachrome isomerase in Drosophila melanogaster.
复制标题
果蝇多巴色素异构酶的电化学鉴定。
DOI：
10.1016/s0006-291x(05)81125-8
复制
发表时间：
1991
影响因子：
3.1
通讯作者：
Nappi,AJ
中科院分区：
文献类型：
--
作者：
Li,J;Nappi,AJ
噬菌体M13外壳蛋白-一种位于大肠杆菌E.在噬菌体繁殖过程中，大肠杆菌宿主膜-在噬菌体生命周期中受到细胞质，膜结合和DNA相互作用环境的影响。在研究中，以检查蛋白质的有效跨膜（TM）区域（残基21-39：YIGYAWAMVVIVGATIGI）的一级/二级结构的具体特征，调节其构象响应这些不同环境的渐进影响的能力，我们已经制备了二十多个在其外壳蛋白TM区域发生改变的可行突变体。通过使用定点诱变技术与跨越TM区域的三种“随机化”寡核苷酸组合获得突变体。不需要亚克隆。在观察到的突变中，有四个TM瓦尔残基中的每一个都变成了Ala，还有几个具有增加的Ser或Thr含量，包括一个双Ser突变体（G23 S-A25 S）。在Gly 23和Tyr 24（包括G23 D、Y24 H、Y24 D和Y24 N）处出现的极性取代表明该局部片段位于宿主膜外部。毫克量的突变体外壳蛋白是通过在升制剂中生长M13突变体大肠杆菌获得的，所述制剂具有同位素（例如，13 C）在所需位点标记，用于随后在膜模拟介质中的表征和构象分析。
Bacteriophage M13 coat protein — a 50-residue protein located at theE. colihost membrane during phage reproduction — is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21–39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three “randomized” oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23and Tyr24— including G23D, Y24H, Y24D and Y24N — suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g.,13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.