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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling1
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Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling1

机译：交联，质谱和组合同源性建模1真核TRiC / CCT伴侣蛋白的亚基顺序1

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The TRiC/CCT chaperonin is a 1-MDa hetero-oligomer of 16 subunits that assists the folding of proteins in eukaryotes. Low-resolution structural studies confirmed the TRiC particle to be composed of two stacked octameric rings enclosing a folding cavity. The exact arrangement of the different proteins in the rings underlies the functionality of TRiC and is likely to be conserved across all eukaryotes. Yet despite its importance it has not been determined conclusively, mainly because the different subunits appear nearly identical under low resolution. This work successfully addresses the arrangement problem by the emerging technique of cross-linking, mass spectrometry, and modeling. We cross-linked TRiC under native conditions with a cross-linker that is primarily reactive toward exposed lysine side chains that are spatially close in the context of the particle. Following digestion and mass spectrometry we were able to identify over 60 lysine pairs that underwent cross-linking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (=8 factorial) computational models of the TRiC particle, which exhaustively enumerate all the possible arrangements of the different subunits. When we assessed the compatibility of each model with the cross-link set, we discovered that one specific model is significantly more compatible than any other model. Furthermore, bootstrapping analysis confirmed that this model is 10 times more likely to result from this cross-link set than the next best-fitting model. Our subunit arrangement is very different than any of the previously reported models and changes the context of existing and future findings on TRiC.
机译：TRiC / CCT伴侣蛋白是16个亚基的1-MDa异源寡聚体，可协助真核生物中蛋白质的折叠。低分辨率的结构研究证实TRiC颗粒由两个折叠的八聚体环组成，它们包围一个折叠腔。环中不同蛋白质的精确排列是TRiC功能的基础，很可能在所有真核生物中都保守。然而，尽管它很重要，但尚未最终确定，主要是因为不同的亚基在低分辨率下看起来几乎相同。这项工作通过新兴的交联，质谱和建模技术成功解决了排列问题。我们在天然条件下用交联剂交联TRiC，该交联剂主要对暴露的赖氨酸侧链具有反应性，而赖氨酸侧链在粒子的背景下在空间上是紧密的。经过消化和质谱分析，我们能够鉴定出60多个进行交联的赖氨酸对，从而在复合物中特定残基之间提供了距离限制。独立于交联集，我们构建了TRiC粒子的40,320（= 8阶乘）计算模型，该模型详尽地枚举了不同亚基的所有可能排列。当我们评估每个模型与交叉链接集的兼容性时，我们发现一个特定的模型比任何其他模型的兼容性都明显更高。此外，自举分析确认该模型比下一个最佳拟合模型产生的交叉链接集高出10倍。我们的子单位安排与以前报告的任何模型都大不相同，并且改变了TRiC现有和将来发现的背景。

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