Fedorov A N, Friguet B, Djavadi-Ohaniance L, Alakhov Y B, Goldberg M E
Institute of Protein Research, Academy of Sciences of Russia Pushchino, Moscow Region.
J Mol Biol. 1992 Nov 20;228(2):351-8. doi: 10.1016/0022-2836(92)90825-5.
Experimental analysis of protein folding during protein synthesis on the ribosome is rendered very difficult by the low concentration of nascent polypeptides and the heterogeneity of the translation mixture. In this study, an original approach is developed for analysing nascent polypeptide structures still carried by the ribosome. Folding on the ribosome of nascent chains of the beta subunit of Escherichia coli tryptophan synthase was investigated using a monoclonal antibody (mAb 19) recognizing a conformation-dependent antigenic determinant. Upon synthesis of beta subunits in an E. coli coupled transcription-translation system, it is shown that ribosome-bound nascent polypeptides can react with the monoclonal antibody provided their size is above 11.5 kDa, which is smaller than that of both the N-terminal proteolytic and crystallographic domains (29 and 21 kDa, respectively). The gene fragments coding only for the 11.5 kDa polypeptide, with and without stop codon at the end of the corresponding mRNAs, were constructed and expressed in a cell-free wheat germ translation system. It is shown that antibody 19 reacts with this polypeptide either bound to the ribosome or free in solution. That the 11.5 kDa polypeptide acquires a condensed structure is shown by gel filtration in native conditions and by urea gradient gel electrophoresis. Moreover, it is demonstrated that this condensed structure resembles that of native beta 2 in the vicinity of the epitope for antibody 19. Indeed, the affinity of antibody 19 for the 11.5 kDa fragment, either free or bound to the ribosome, was measured (6 x 10(8) M-1) and shown to be close to that for native beta 2. It is therefore proposed that the polypeptide chain may start to fold during its biosynthesis and that, even before the appearance of an entire domain, a folded intermediate is formed that already exhibits some local structural features of the native state and of an immunoreactive intermediate previously detected during the in vitro refolding of denatured complete beta chains.
核糖体上蛋白质合成过程中蛋白质折叠的实验分析，因新生多肽浓度低以及翻译混合物的异质性而变得极为困难。在本研究中，开发了一种原始方法来分析仍由核糖体携带的新生多肽结构。使用识别构象依赖性抗原决定簇的单克隆抗体（mAb 19），研究了大肠杆菌色氨酸合酶β亚基新生链在核糖体上的折叠情况。在大肠杆菌偶联转录 - 翻译系统中合成β亚基时，结果表明，核糖体结合的新生多肽只要其大小超过11.5 kDa，就能与单克隆抗体发生反应，该大小比N端蛋白水解结构域和晶体结构域（分别为29 kDa和21 kDa）都小。构建了仅编码11.5 kDa多肽的基因片段，其相应mRNA末端有或无终止密码子，并在无细胞小麦胚芽翻译系统中表达。结果表明，抗体19能与结合在核糖体上或游离于溶液中的该多肽发生反应。在天然条件下的凝胶过滤和尿素梯度凝胶电泳表明，11.5 kDa多肽获得了一种浓缩结构。此外，还证明这种浓缩结构类似于抗体19表位附近天然β2的结构。实际上，测量了抗体19对游离或结合在核糖体上的11.5 kDa片段的亲和力（6×10⁸ M⁻¹），结果表明其与对天然β2的亲和力相近。因此，有人提出多肽链可能在其生物合成过程中就开始折叠，并且即使在整个结构域出现之前，就已形成一种折叠中间体，该中间体已经展现出天然状态以及先前在变性完整β链的体外重折叠过程中检测到的免疫反应性中间体的一些局部结构特征。
