Yokohama City University, Supramolecular Biology, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Anal Chem. 2009 Mar 15;81(6):2218-26. doi: 10.1021/ac802354j.
We have characterized both wild-type and mutant TRAP (trp RNA-binding attenuation protein) from Bacillus stearothermophilus , and their complexes with RNA or its regulator anti-TRAP protein (AT), by electrospray ionization mass spectrometry (ESI-MS). Wild-type TRAP mainly forms homo-11mer rings. The mutant used carries three copies of the TRAP monomer on a single polypeptide chain so that it associates to form a 12mer ring with four polypeptide molecules. Mass spectra showed that both the wild-type TRAP 11mer and the mutant TRAP 12mer can bind a cognate single-stranded RNA molecule with a molar ratio of 1:1. The crystal structure of wild-type TRAP complexed with AT shows a TRAP 12mer ring surrounded by six AT trimers. However, nanoESI-MS of wild-type TRAP mixed with AT shows four species with different binding stoichiometries, and the complex observed by crystallography represents only a minor species in solution; most of the TRAP remains in an 11mer ring form. Mass spectra of mutant TRAP showed only a single species, TRAP 12mer + six copies of AT trimer, which is observed by crystallography. These results suggest that crystallization selects only the most symmetrical TRAP-AT complex from the solution, whereas ESI-MS can take a "snapshot" of all the species in solution.
我们通过电喷雾电离质谱（ESI-MS）对来自嗜热脂肪芽孢杆菌的野生型和突变型 TRAP（色氨酸 RNA 结合衰减蛋白）及其与 RNA 或其调节剂抗 TRAP 蛋白（AT）的复合物进行了表征。野生型 TRAP 主要形成同型 11 聚体环。所用的突变体在单个多肽链上携带三个 TRAP 单体拷贝，因此它可以与四个多肽分子缔合形成具有 12 个单体的环。质谱表明，野生型 TRAP 11 聚体和突变型 TRAP 12 聚体都可以与同源的单链 RNA 分子以 1:1 的摩尔比结合。与 AT 结合的野生型 TRAP 的晶体结构显示 TRAP 12 聚体环被六个 AT 三聚体包围。然而，与 AT 混合的野生型 TRAP 的纳喷雾电离质谱显示出具有不同结合化学计量比的四种物质，并且晶体学观察到的复合物仅代表溶液中的次要物质；大部分 TRAP 仍以 11 聚体环的形式存在。突变型 TRAP 的质谱仅显示一种物质，即 TRAP 12 聚体+六个 AT 三聚体，这是晶体学观察到的。这些结果表明，结晶仅从溶液中选择最对称的 TRAP-AT 复合物，而 ESI-MS 可以对溶液中的所有物质进行“快照”。
