MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics.
MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics.
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DOI：
10.1016/j.mcpro.2022.100256
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发表时间：
2022-07
影响因子：
7
通讯作者：
Londino, James D.
中科院分区：
文献类型：
--
作者：
Johnson, Benjamin S.;Chafin, Lexie;Farkas, Daniela;Adair, Jessica;Elhance, Ajit;Farkas, Laszlo;Bednash, Joseph S.;Londino, James D.
识别蛋白质-蛋白质和其他近端相互作用是解剖细胞中信号传导和调控过程的核心。BioID是一种邻近依赖性生物素化方法，其使用“流产”生物素连接酶以高度可重复的方式检测细胞中的邻近相互作用。邻近依赖性生物素化工具的最新进展提高了标记的效率和时间，允许在细胞时间尺度上测量相互作用。然而，这些结构的大小、稳定性和背景标记的问题仍然存在。在这里，我们修改了来自Aquifex aeolicus BirA的BioID 2的结构，以创建一种更小，高活性的生物素连接酶，我们将其命名为MicroID 2。截短BioID 2的C末端并添加突变以减轻BioID 2活性位点处生物素/ATP结合的阻断，导致具有较低背景标记的较小且高活性的构建体。与BioID 2和其他生物素连接酶（包括TurboID和miniTurbo）相比，几个额外的点突变改善了我们修饰的MicroID 2构建体的功能。MicroID 2是迄今为止报道的最小的生物素连接酶（MicroID 2为180个氨基酸[AA]，miniTurbo为257个AA，TurboID为338个AA），但它的标记活性仅略低于TurboID，并且优于miniTurbo。MicroID 2的背景标记也低于TurboID。对于标记的精确时间控制至关重要的实验，我们另外开发了一种MicroID 2突变体，称为lbMicroID 2（低背景MicroID 2），其与BioID 2相比具有较低的标记效率但显著降低了生物素清除。最后，我们展示了MicroID 2在质谱实验中的实用性，通过将MicroID 2构建体定位到亚细胞器并测量近端相互作用。MicroID 2是迄今为止描述的最小的生物素连接酶，其促进近端蛋白质的稳健标记。低背景MicroID 2允许较慢的标记，具有较少的背景生物素化。MicroID 2可用作亚细胞器中邻近蛋白质组学的工具。BioID使用混杂的生物素连接酶以高度可重复的方式检测蛋白质-蛋白质相互作用。这些结构的大小、稳定性和背景标签问题仍然存在。在这里，我们修改了BioID 2的结构以创建MicroID 2，这是所描述的最小的生物素连接酶。MicroID 2具有与TurboID相当的标签，并且优于miniTurbo。我们还开发了lbMicroID 2，一种具有较低标记和较少背景的突变体。最后，我们通过测量检测亚细胞器中的蛋白质，证明了MicroID 2在质谱中的实用性。
Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions. MicroID2 is smallest biotin ligase yet described that facilitates robust labeling of proximal proteins. Low background MicroID2 allows slower labeling with less background biotinylation. MicroID2 can be utilized as a tool for proximity proteomics in subcellular organelles. BioID uses a promiscuous biotin ligase to detect protein–protein interactions in a highly reproducible manner. Issues of size, stability, and background labeling of these constructs persist. Here, we modified the structure of BioID2 to create MicroID2, the smallest biotin ligase described. MicroID2 has comparable labeling to TurboID and outperforms miniTurbo. We also developed lbMicroID2, a mutant with lower labeling and less background. Finally, we demonstrated the utility of MicroID2 in mass spectrometry by measuring examining proteins in subcellular organelles.
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DOI：
10.1093/bioinformatics/btw580
发表时间：
2017-01-01
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Wieczorek S;Combes F;Lazar C;Giai Gianetto Q;Gatto L;Dorffer A;Hesse AM;Couté Y;Ferro M;Bruley C;Burger T
通讯作者：
Burger T
影响因子：
7.7
作者：
Hung V;Lam SS;Udeshi ND;Svinkina T;Guzman G;Mootha VK;Carr SA;Ting AY
通讯作者：
Ting AY
DOI：
10.1016/j.mcpro.2021.100186
发表时间：
2022-03
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Christopher JA;Geladaki A;Dawson CS;Vennard OL;Lilley KS
通讯作者：
Lilley KS
DOI：
10.1007/978-1-4939-9546-2_15
发表时间：
2019-01-01
期刊：
ENZYME-MEDIATED LIGATION METHODS
影响因子：
--
作者：
Sears, Rhiannon M.;May, Danielle G.;Roux, Kyle J.
通讯作者：
Roux, Kyle J.
影响因子：
6
作者：
May, Danielle G.;Scott, Kelsey L.;Roux, Kyle J.
通讯作者：
Roux, Kyle J.