Fast Biosynthesis of GFP Molecules: A Single-Molecule Fluorescence Study
Fast Biosynthesis of GFP Molecules: A Single-Molecule Fluorescence Study
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DOI：
10.1002/anie.200806070
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Fitter, Joerg
中科院分区：
文献类型：
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作者：
Katranidis, Alexandros;Atta, Diaa;Fitter, Joerg
大量研究表明，从头合成的蛋白质和体外重折叠的蛋白质之间的蛋白质折叠和成熟可能存在很大差异。 [1-3] 在经典折叠研究中，在研究折叠（或更确切地说，重折叠）过程之前，需要将先前折叠的蛋白质转移到未折叠状态。多个案例已证明蛋白质折叠在新生链伸长过程中已经发生（共翻译折叠）。蛋白质可以完全折叠并具有酶活性，同时仍然通过跨越核糖体通道的约 30 个氨基酸的 C 端延伸与核糖体结合。[4-7] 在折叠率、折叠中间体的出现和产量方面，观察到从头合成蛋白质的折叠和体外重折叠之间存在显着差异。[2,8,9]因此，一个主要目标是了解多肽链延伸和折叠是如何进行的耦合。特别是，单分子研究可以产生有关这些相当异步的过程的有价值的信息。核糖体复合物作为将遗传密码信息转换为多肽链的机器，已经通过各种单分子技术进行了研究。[10-14]在这里，我们在从头合成和折叠后在单分子水平上观察到绿色荧光蛋白（GFP）。荧光发色团的形成是一个相当缓慢的翻译后自催化过程，并且 GFP 野生型和几种突变体之间的成熟动力学和折叠效率存在显着差异。 [15]我们选择了 GFP Emerald (GFPem) 突变体，其特点是高折叠效率以及快速折叠和成熟动力学。 [16, 17] 使用分级分离的无细胞转录-翻译大肠杆菌系统完成表面固定荧光标记核糖体上的 GFP 合成（图 1）。 GFPem的序列被拉长
Numerous studies have shown that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins.[1–3] In classical folding studies, formerly folded proteins need to be transferred into an unfolded state before the folding (or rather, refolding) process can be studied. It has been demonstrated in several cases that protein folding already takes place during the elongation of the nascent chain (cotranslational folding). Proteins can become fully folded and enzymatically active while they are still bound to the ribosome through a C-terminal extension of about 30 amino acids that spans the ribosomal channel.[4–7] Significant differences have been observed between folding of de novo synthesized proteins and in vitro refolding with respect to folding rates, the appearance of folding intermediates, and yields.[2, 8, 9] Therefore, one major goal is to understand how polypeptide chain elongation and folding are coupled. In particular, singlemolecule studies can yield valuable information about these rather asynchronous processes. The ribosomal complex as a machine converting the information of the genetic code into a polypeptide chain has already been studied with various single-molecule techniques.[10–14]Herein, we observed green fluorescent proteins (GFPs) at a single-molecule level after de novo synthesis and folding. Formation of the fluorescent chromophore is a rather slow post-translational autocatalytic process, and the maturation kinetics as well as the folding efficiency differ significantly between GFP wild type and several mutants.[15] We have chosen the GFP Emerald (GFPem) mutant, which is characterized by a high folding efficiency and by fast folding and maturation kinetics.[16, 17] GFP synthesis at surface-immobilized fluorescently labeled ribosomes was accomplished using a fractionated cell-free transcription–translation E. coli system (Figure 1). The sequence of GFPem was elongated