Antiparallel leucine zipper-directed protein reassembly: Application to the green fluorescent protein
Antiparallel leucine zipper-directed protein reassembly: Application to the green fluorescent protein
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DOI：
10.1021/ja994421w
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发表时间：
2000-06-14
影响因子：
15
通讯作者：
Regan, L
中科院分区：
文献类型：
--
作者：
Ghosh, I;Hamilton, AD;Regan, L
蛋白质从肽片段中的分离和随后的重组为控制其三级结构和功能提供了一条途径。在这里，我们描述了一个通用的方法，重组蛋白质片段介导的非共价关联的反平行亮氨酸拉链。[1]虽然大多数亮氨酸拉链以平行方式缔合，但最近的文献中出现了天然存在的和设计的反平行亮氨酸拉链的例子。1，2我们在这里报告了一种通过反平行亮氨酸拉链将蛋白质切割表面环的N-和C-末端非共价重连接的策略（图1）。3我们已经成功地将这种寡聚化策略应用于来自维多利亚水母的238个残基的绿色荧光蛋白（GFP）的体外和体内。4 GFP提供了一个易于测试的系统，用于正确的重组，凭借其自催化产生的荧光，这是密切联系到其正确的折叠结构。5此外，目前对利用GFP作为生物传感器的兴趣为基于我们描述的策略产生用于生物技术应用的新工具提供了进一步的动机。[5]对于几种蛋白质，包括核糖核酸酶、6a胰凝乳蛋白酶抑制剂-2、6 b tRNA合成酶、6c和内含肽，已经证明了从肽片段无辅助地重建蛋白质。因此，6d蛋白质重组已成为理解酶催化、6a蛋白质折叠、6 b和蛋白质进化的重要途径。6c最近，辅助蛋白质重组或“片段互补”已被应用于体内检测诸如二氢叶酸还原酶（DHFR）、7a-c泛素、7 d、e和β-半乳糖苷酶等系统中的蛋白质-蛋白质相互作用。这些重组过程取决于蛋白质内切割位点的正确选择，并可通过有限蛋白水解、环状置换8和环插入等技术来辅助。9特别是，最近的环状排列8 c和蛋白质插入8b，9策略提供了令人信服的证据，表明GFP可以折叠，发荧光，并作为生物传感器，尽管天然编码序列的重排。在我们的研究中，我们使用了天然存在的GFP的变体，其在475 nm处具有单一激发最大值。10我们的设计策略要求在残基157和158之间的表面环处切割GFP，该位置先前已被证明可容纳20个残基的氨基酸插入。9a我们的解剖产生了N-和C-末端片段，命名为NGFP和CGFP，分别含有157和81个残基（图1）。NGFP片段含有三个残基，Ser 65，Tyr 66和Gly 67，它们最终形成GFP荧光团。[4]为了重组目的，设计螺旋（命名为NZ和CZ）以形成反平行亮氨酸拉链是基于Hodges，11 a Kim，11 b和阿尔伯报道的序列。11 c亮氨酸拉链含有富含Leu的疏水性核心、酸性（Glu）和碱性（Lys）残基，以指导反平行异二聚体的形成，并且还包含一个埋藏的天冬酰胺残基，该残基不利于同二聚化高达2.3 kcal/mol（图1）。2a将设计的螺旋NZ通过6个残基的接头附加到NGFP的C-末端，以产生指定的片段
The dissection and subsequent reassembly of a protein from peptidic fragments provides an avenue for controlling its tertiary structure and hence its function. Here, we describe a general method for the reassembly of protein fragments mediated by the noncovalent association of antiparallel leucine zippers. 1 Although a majority of leucine zippers associate in a parallel fashion, recent examples of both naturally occurring and designed antiparallel leucine zippers have appeared in the literature. 1, 2 We report here a strategy for the noncovalent reconnection of the N-and C-termini of a dissected surface loop of a protein by means of antiparallel leucine zippers (Figure 1). 3 We have successfully applied this oligomerization strategy, both in vitro and in vivo, to the 238 residue green fluorescent protein (GFP) from Aequorea Victoria. 4 GFP provides an easily testable system for correct reassembly by virtue of its autocatalytically generated fluorescence, which is intimately linked to its properly folded structure. 5 Moreover, the current interest in utilizing GFP as a biosensor provides further motivation for generating new tools for biotechnological applications based on the strategy we describe. 5 The unassisted reconstitution of proteins from peptide fragments has been demonstrated for several proteins; including ribonuclease, 6a chymotrypsin inhibitor-2, 6b tRNA synthetases, 6c and inteins. 6d Protein reassembly has thus become an important avenue for understanding enzyme catalysis, 6a protein folding, 6b and protein evolution. 6c Recently, assisted protein reassembly or “fragment complementation” has been applied to the in vivo detection of protein-protein interactions in such systems as dihydrofolate reductase (DHFR), 7a-c ubiquitin, 7d, e and β-galactosidase. 7f These reassembly processes are contingent upon the proper choice of a dissection site within a protein and can be aided by techniques such as limited proteolysis, circular permutation8 and loop insertions. 9 In particular, recent circular permutation8c and protein insertion8b, 9 strategies have provided convincing evidence that GFP can fold, fluoresce, and serve as a biosensor despite the rearrangement of the natural coding sequence. In our study we have used a variant of the naturally occurring GFP, which has a single excitation maximum at 475 nm. 10 Our design strategy called for the dissection of GFP at a surface loop between residues 157 and 158, a position that has previously been shown to accommodate a 20-residue amino acid insertion. 9a Our dissection resulted in N-and C-terminal fragments, designated NGFP and CGFP, containing 157 and 81 residues, respectively (Figure 1). The NGFP fragment contains the three residues, Ser65, Tyr66, and Gly67, that ultimately form the GFP fluorophore. 4 Designs for helices, designated NZ and CZ, to form antiparallel leucine zippers for reassembly purposes were based upon sequences reported by Hodges, 11a Kim, 11b and Alber. 11c The leucine zippers contained a Leu-rich hydrophobic core, acidic (Glu) and basic (Lys) residues to direct antiparallel heterodimer formation, and also incorporated a buried asparagine residue which disfavors homodimerization by up to 2.3 kcal/mol (Figure 1). 2a The designed helix, NZ was appended to the C-terminal of NGFP, via a 6-residue linker, to generate the fragment designated