Folding of large multidomain proteins by partial encapsulation in the chaperonin TRiC/CCT
Folding of large multidomain proteins by partial encapsulation in the chaperonin TRiC/CCT
复制标题
DOI：
10.1073/pnas.1218836109
复制
发表时间：
2012-12-26
影响因子：
11.1
通讯作者：
Hartl, F. Ulrich
中科院分区：
文献类型：
--
作者：
Ruessmann, Florian;Stemp, Markus J.;Hartl, F. Ulrich
真核分子伴侣蛋白TRiC/CCT（TRiC，TCP-1环复合物; CCT，含有TCP-1的分子伴侣蛋白）使用内置的盖子来介导封闭的中心腔中的蛋白质折叠。最近的结构数据表明，TRiC折叠室的有效大小限制类似于70 kDa，但许多伴侣蛋白底物大得多。使用人工融合结构与肌动蛋白，一个专性伴侣蛋白底物，我们表明，TRiC可以介导折叠的大蛋白质的节段性或结构域的封装。通过稳定TRiC的ATP-水解过渡态，可稳定封闭高达70 kDa的单个或多个蛋白质结构域。通过穿过折叠室的中心开口的柔性接头连接的另外的结构域被排除，并且保持可接近外部添加的蛋白酶。实验与TRiC的生理底物hSnu 114，109 kDa的多结构域蛋白，表明TRiC有能力识别结构域边界的部分折叠的中间体。在hSnu 114的情况下，这允许选择性包封C-末端类似于45-kDa结构域及其片段，推测反映了逐步折叠机制。真核生物伴侣蛋白克服折叠室大小限制的能力可能促进了真核生物多结构域蛋白质组的爆炸性扩张。
The eukaryotic chaperonin, TRiC/CCT (TRiC, TCP-1 ring complex; CCT, chaperonin containing TCP-1), uses a built-in lid to mediate protein folding in an enclosed central cavity. Recent structural data suggest an effective size limit for the TRiC folding chamber of similar to 70 kDa, but numerous chaperonin substrates are substantially larger. Using artificial fusion constructs with actin, an obligate chaperonin substrate, we show that TRiC can mediate folding of large proteins by segmental or domain-wise encapsulation. Single or multiple protein domains up to similar to 70 kDa are stably enclosed by stabilizing the ATP-hydrolysis transition state of TRiC. Additional domains, connected by flexible linkers that pass through the central opening of the folding chamber, are excluded and remain accessible to externally added protease. Experiments with the physiological TRiC substrate hSnu114, a 109-kDa multidomain protein, suggest that TRiC has the ability to recognize domain boundaries in partially folded intermediates. In the case of hSnu114, this allows the selective encapsulation of the C-terminal similar to 45-kDa domain and segments thereof, presumably reflecting a stepwise folding mechanism. The capacity of the eukaryotic chaperonin to overcome the size limitation of the folding chamber may have facilitated the explosive expansion of the multidomain proteome in eukaryotes.