Subcellular protein localization by using a genetically encoded fluorescent amino acid.
Subcellular protein localization by using a genetically encoded fluorescent amino acid.
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使用基因编码的荧光氨基酸进行亚细胞蛋白质定位
DOI：
10.1002/cbic.201100282
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发表时间：
2011-08-16
期刊：
影响因子：
3.2
通讯作者：
Chapman, Eli
中科院分区：
文献类型：
--
作者：
Charbon, Godefroid;Brustad, Eric;Scott, Kevin A.;Wang, Jiangyun;Lobner-Olesen, Anders;Schultz, Peter G.;Jacobs-Wagner, Christine;Chapman, Eli
荧光蛋白融合体的使用通过允许在其天然环境中探索蛋白质而彻底改变了细胞生物学。然而，目前技术的利用受到融合荧光蛋白的大小和位置的限制。对于寡聚化或组装成大复合物的蛋白质尤其如此，其中荧光蛋白融合可导致不适当的组装和/或功能。为了解决这个问题，我们报告了一种新的技术，在体内的蛋白质的荧光标记。该技术的独特潜力在于能够将非常小的荧光标记物放置在所选蛋白质序列的几乎任何沿着，[1，2]从而最小化影响蛋白质功能的风险。为了说明这种方法的实用性，我们在细菌微管蛋白FtsZ的序列中遗传编码了一个非天然的荧光氨基酸。这导致了可以在体内可视化的功能性蛋白质的产生。本研究的目的是探索新的方法标记蛋白质在体内定位研究，不干扰蛋白质的功能或结构。为此，我们开发了一种技术，允许具有新特性的非天然氨基酸，在我们的情况下，荧光香豆素衍生的氨基酸（CouAA;图1A），被编码到给定的序列中。[1，2]该技术使用正交tRNA/氨酰-tRNA合成酶对，当编码序列中存在无义琥珀密码子（UAG）[1]时，该合成酶将定义的非天然氨基酸转移到生长的多肽链。具体而言，我们使用了古细菌琥珀抑制tRNA（MjtRNA）/氨酰-tRNA合成酶（Mj-aaRS）对（来自詹氏甲烷球菌），其不与内源性E.大肠杆菌氨酰-tRNA合成酶或tRNA。[1]该阿尔斯（CouRS）然后通过使用两步选择过程来进化，以特异性识别CouAA而不是内源性宿主氨基酸。因此，通过简单地在所需基因序列中插入琥珀终止密码子，将在相应的翻译蛋白质中引入非天然氨基酸（参见支持信息中的漫画）。方便地，E.大肠杆菌和其他生物体很可能不被Mj-tRNA识别，因此由琥珀密码子终止的基因的表达显然不受影响。虽然确切的机制还不清楚，但已知终止和抑制过程受到琥珀密码子附近序列的影响。[3-6]为了证明该系统可以有效地用作使CouAA标记的蛋白质在细菌中的体内亚细胞位置可视化的手段，我们选择标记细菌微管蛋白同源物FtsZ，其已经在体外和体内进行了广泛的研究。在胞质分裂期间，FtsZ组装成收缩环（通过荧光显微镜可见，作为称为Z环的中细胞带）。[7-9]但是FtsZ与荧光蛋白的融合会损害其细胞功能，这是细胞骨架蛋白的一个众所周知的问题。因此，迄今为止，所有报道的FtsZ-荧光蛋白融合体已被证明是非功能性的本身，必须在一个未标记的FtsZ拷贝的存在下产生的正常细胞功能。[10]因此，在这种情况下，FtsZ-荧光蛋白融合体仅标记内源性的未标记的FtsZ结构。虽然这种方法已被证明足以对FtsZ细胞功能产生相当大的洞察力，但它仍然是一种不完美的技巧，在其他寡聚体形成蛋白或蛋白质形成大分子复合物的情况下可能会失败。在E.在大肠杆菌中，我们替换了第十个氨基酸...
The use of fluorescent protein fusions has revolutionized cell biology by allowing exploration of proteins in their native context. However, the utilization of current techniques is limited by the size and placement of the fused fluorescent protein. This is especially true for proteins that oligomerize or assemble into large complexes in which the fluorescent protein fusion can lead to improper assembly and/or function. To circumvent this problem, we report a novel technique for fluorescent labeling of proteins, in vivo. The unique potential of this technique lies in the ability to place a very small fluorescent tag virtually anywhere along a chosen protein sequence,[1, 2] thereby minimizing the risk of affecting protein function. To illustrate the utility of this method, we have genetically encoded a single unnatural fluorescent amino acid in the sequence of the bacterial tubulin, FtsZ. This resulted in the production of a functional protein that could be visualized, in vivo. The aim of this study was to explore novel methods of labeling proteins for in vivo localization studies that do not perturb protein function or structure. To this end, we have exploited a technique that allows unnatural amino acids with novel properties, in our case a fluorescent, coumarin-derived amino acid (CouAA; Figure 1A), to be encoded into a given sequence.[1, 2] The technique uses an orthogonal tRNA/aminoacyl-tRNA synthetase pair that transfers a defined unnatural amino acid to a growing polypeptide chain when nonsense amber codons (UAG)[1] are present in the coding sequence. Specifically, we have used an archaebacteria amber suppressor tRNA (MjtRNA)/aminoacyl-tRNA synthetase (Mj-aaRS) pair (from Methanococcus jannaschi) that does not interact with endogenous E. coli aminoacyl-tRNA synthetases or tRNAs.[1] This aaRS (CouRS) was then evolved by using a two-step selection process to specifically recognize CouAA and not an endogenous host amino acid. As a consequence, by simply inserting an amber stop codon in the desired gene sequence, an unnatural amino acid will be introduced in the corresponding translated protein (see cartoon in the Supporting Information). Conveniently, endogenous amber codons in E. coli and other organisms are likely poorly recognized by the Mj-tRNA, so that the expression of genes terminated by amber codons is apparently not affected. Although the exact mechanisms are not understood, it is known that the termination and suppression processes are influenced by sequences adjacent to the amber codon.[3–6]To demonstrate that this system can be effectively used as a means to visualize the in vivo subcellular location of a CouAA-labeled protein in bacteria, we chose to label the bacterial tubulin homologue FtsZ, which has been extensively studied both in vitro and in vivo. FtsZ assembles into a contractile ring (visible by fluorescence microscopy as a midcell band called Z-ring) during cytokinesis.[7–9] But fusion of FtsZ to a fluorescent protein impairs its cellular function, a well-known problem for cytoskeletal proteins. Consequently, to date, all the reported FtsZ–fluorescent protein fusions have been shown to be nonfunctional on their own and must be produced in the presence of an untagged FtsZ copy for normal cell function.[10] Thus, in this context, the FtsZ–fluorescent protein fusions merely label the endogenous, untagged FtsZ structure. While this approach has proven to be sufficient to generate considerable insight into FtsZ cellular function, it remains an imperfect artifice that might fail in the case of other oligomer-forming proteins or proteins forming macromolecular complexes. To visualize FtsZ in E. coli, we substituted the tenth amino acid …
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DOI：
10.1073/pnas.93.23.12998
发表时间：
1996-11-12
影响因子：
11.1
作者：
Ma, XL;Ehrhardt, DW;Margolin, W
通讯作者：
Margolin, W
影响因子：
64.5
作者：
WARD, JE;LUTKENHAUS, J
通讯作者：
LUTKENHAUS, J
影响因子：
5.6
作者：
Young, Travis S.;Ahmad, Insha;Schultz, Peter G.
通讯作者：
Schultz, Peter G.
影响因子：
3.2
作者：
Anderson, DE;Gueiros-Filho, FJ;Erickson, HP
通讯作者：
Erickson, HP
影响因子：
64.5
作者：
Ben-Yehuda, S;Losick, R
通讯作者：
Losick, R