The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence...The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence intensity of fluorescein dimercury acetate(FDMA)(in 1 mol/L NaOH) were investigated by fluorophotometric titrations and compared with those of (NH 4) 2MoS 4 and complexes of (NH 4) 2MoS 4 with Na 2S or Na 2S 2 or (NH 4) 2S x on the relative properties of FDMA. It was found that the electronic spectrum of FDMA displayed hypsochromic shift(17 nm) in the presence of FeMoco just like that in the presence of other inorganic sulfides and that the titration curve for the quench of FDMA with FeMoco is very similar to that for the quench of FDMA with complex of (NH 4) 2MoS 4 with Na 2S 2(mole ratio is 1∶3). The results showed that FeMoco(N) probably contained S—S bonds and its structure was found to be changed compared with Kim Rees structural model. This change should profit the formation of the polymer.展开更多
【目的】微小杆菌属(Exiguobacterium)细菌广泛分布于海洋及非海洋环境中,具有多种代谢途径以适应复杂多样的生境。本研究从能量代谢途径角度出发,探究该属菌株对不同生境的适应能力。【方法】从美国国家生物科技数据中心(National Cent...【目的】微小杆菌属(Exiguobacterium)细菌广泛分布于海洋及非海洋环境中,具有多种代谢途径以适应复杂多样的生境。本研究从能量代谢途径角度出发,探究该属菌株对不同生境的适应能力。【方法】从美国国家生物科技数据中心(National Center for Biotechnology Information, NCBI)数据库中获取146个Exiguobacterium属菌株的基因组,查找并统计光营养、厌氧呼吸和底物代谢等多种能量代谢途径的关键蛋白或关键酶基因在各菌株基因组中的分布,包括光营养型的视紫红质基因、厌氧呼吸营养型的钼辅因子合成蛋白基因,以及底物代谢营养型中乙醛酸分流途径的异柠檬酸裂解酶及苹果酸合酶基因等。根据对应的氨基酸序列构建视紫红质、MoaC和异柠檬酸裂解酶的系统发育树,分析不同能量代谢途径在该属菌株进化过程中的保守性,推测其对于该属菌株的重要性。【结果】Exiguobacterium属中50%的种具有视紫红质基因,其中分离自非海洋生境的菌株更趋向于含有视紫红质基因。本研究所统计的全部非海洋生境菌株中,含有视紫红质基因的菌株占比约为70%,而在海洋生境菌株中该比例仅为19%。Exiguobacterium属约27%的种存在钼辅因子合成蛋白基因,分离自海洋环境的菌株有该类蛋白的可能性更高(32%:21%),具有完整钼辅因子合成途径的菌株集中于进化树同一分支上的少数种。该属约61%的种存在乙醛酸分流途径相关酶基因,这些种隶属进化树的同一分支,且种内所有菌株都具备相关基因,表明乙醛酸分流途径在Exiguobacterium属的分布具有种特异性。【结论】Exiguobacterium属细菌具有多种能量代谢途径相关基因,包括基于视紫红质的光营养型能量代谢途径、基于钼酶的厌氧呼吸型能量代谢途径和底物代谢营养型能量代谢途径中的乙醛酸分流途径。能量代谢途径多样性可能是Exiguobacterium属细菌适应复杂多样生境的机制之一。此外,本研究发现Exiguobacterium属中不同种、同种不同菌株间存在能量代谢途径差异,且能量代谢途径在该属内的分布多不具备种特异性,表明仅通过16S rRNA基因种属鉴定来预测目标菌株的代谢类型可能有较大偏差和局限性。展开更多
Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and th...Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.展开更多
An ever-increasing number of intracellular multi-protein networks have been identified in plant cells.Split-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a nati...An ever-increasing number of intracellular multi-protein networks have been identified in plant cells.Split-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization.Because of their simple protocols,they have become some of the most frequently used methods.However,standard fluorescent proteins present several drawbacks for sophisticated microscopy.With the HaloTag system,these drawbacks can be overcome,as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands.Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods.Therefore,we have established the Split-HaloTag imaging assay in plants,which is based on the reconstitution of a functional HaloTag protein upon protein–protein interaction and the subsequent covalent binding of an added fluorescent ligand.Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein–protein interaction at cellular structures:the anchoring of the molybdenumcofactor biosynthesis complex to filamentous actin.In addition,a specific interactionwas visualized in a more distinctivemannerwith subdiffractional polarizationmicroscopy,Airyscan,and structured illumination microscopy to provide examples of sophisticated imaging.Split-GFPand Split-HaloTag can complement one another,as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods.Therefore,this promising new Split-HaloTag imaging assay provides a unique and sensitive approach formore detailed characterization of protein–protein interactions using specific microscopy techniques,such as 3D imaging,single-molecule tracking,and super-resolution microscopy.展开更多
文摘The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence intensity of fluorescein dimercury acetate(FDMA)(in 1 mol/L NaOH) were investigated by fluorophotometric titrations and compared with those of (NH 4) 2MoS 4 and complexes of (NH 4) 2MoS 4 with Na 2S or Na 2S 2 or (NH 4) 2S x on the relative properties of FDMA. It was found that the electronic spectrum of FDMA displayed hypsochromic shift(17 nm) in the presence of FeMoco just like that in the presence of other inorganic sulfides and that the titration curve for the quench of FDMA with FeMoco is very similar to that for the quench of FDMA with complex of (NH 4) 2MoS 4 with Na 2S 2(mole ratio is 1∶3). The results showed that FeMoco(N) probably contained S—S bonds and its structure was found to be changed compared with Kim Rees structural model. This change should profit the formation of the polymer.
文摘【目的】微小杆菌属(Exiguobacterium)细菌广泛分布于海洋及非海洋环境中,具有多种代谢途径以适应复杂多样的生境。本研究从能量代谢途径角度出发,探究该属菌株对不同生境的适应能力。【方法】从美国国家生物科技数据中心(National Center for Biotechnology Information, NCBI)数据库中获取146个Exiguobacterium属菌株的基因组,查找并统计光营养、厌氧呼吸和底物代谢等多种能量代谢途径的关键蛋白或关键酶基因在各菌株基因组中的分布,包括光营养型的视紫红质基因、厌氧呼吸营养型的钼辅因子合成蛋白基因,以及底物代谢营养型中乙醛酸分流途径的异柠檬酸裂解酶及苹果酸合酶基因等。根据对应的氨基酸序列构建视紫红质、MoaC和异柠檬酸裂解酶的系统发育树,分析不同能量代谢途径在该属菌株进化过程中的保守性,推测其对于该属菌株的重要性。【结果】Exiguobacterium属中50%的种具有视紫红质基因,其中分离自非海洋生境的菌株更趋向于含有视紫红质基因。本研究所统计的全部非海洋生境菌株中,含有视紫红质基因的菌株占比约为70%,而在海洋生境菌株中该比例仅为19%。Exiguobacterium属约27%的种存在钼辅因子合成蛋白基因,分离自海洋环境的菌株有该类蛋白的可能性更高(32%:21%),具有完整钼辅因子合成途径的菌株集中于进化树同一分支上的少数种。该属约61%的种存在乙醛酸分流途径相关酶基因,这些种隶属进化树的同一分支,且种内所有菌株都具备相关基因,表明乙醛酸分流途径在Exiguobacterium属的分布具有种特异性。【结论】Exiguobacterium属细菌具有多种能量代谢途径相关基因,包括基于视紫红质的光营养型能量代谢途径、基于钼酶的厌氧呼吸型能量代谢途径和底物代谢营养型能量代谢途径中的乙醛酸分流途径。能量代谢途径多样性可能是Exiguobacterium属细菌适应复杂多样生境的机制之一。此外,本研究发现Exiguobacterium属中不同种、同种不同菌株间存在能量代谢途径差异,且能量代谢途径在该属内的分布多不具备种特异性,表明仅通过16S rRNA基因种属鉴定来预测目标菌株的代谢类型可能有较大偏差和局限性。
文摘Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.
基金supported by the Deutsche Forschungsgemeinschaft(grant GRK2223/1)to R.H.and R.R.M.
文摘An ever-increasing number of intracellular multi-protein networks have been identified in plant cells.Split-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization.Because of their simple protocols,they have become some of the most frequently used methods.However,standard fluorescent proteins present several drawbacks for sophisticated microscopy.With the HaloTag system,these drawbacks can be overcome,as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands.Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods.Therefore,we have established the Split-HaloTag imaging assay in plants,which is based on the reconstitution of a functional HaloTag protein upon protein–protein interaction and the subsequent covalent binding of an added fluorescent ligand.Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein–protein interaction at cellular structures:the anchoring of the molybdenumcofactor biosynthesis complex to filamentous actin.In addition,a specific interactionwas visualized in a more distinctivemannerwith subdiffractional polarizationmicroscopy,Airyscan,and structured illumination microscopy to provide examples of sophisticated imaging.Split-GFPand Split-HaloTag can complement one another,as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods.Therefore,this promising new Split-HaloTag imaging assay provides a unique and sensitive approach formore detailed characterization of protein–protein interactions using specific microscopy techniques,such as 3D imaging,single-molecule tracking,and super-resolution microscopy.
