【目的】验证根癌农杆菌(Agrobacterium fabrum,以前也叫Agrobacterium tumefaciens)两种储铁蛋白——饥饿细胞的DNA结合蛋白(DNA-binding protein from starved cells, Dps)和细菌铁蛋白(bacterioferritin, Bfr)的编码基因atu2477和atu...【目的】验证根癌农杆菌(Agrobacterium fabrum,以前也叫Agrobacterium tumefaciens)两种储铁蛋白——饥饿细胞的DNA结合蛋白(DNA-binding protein from starved cells, Dps)和细菌铁蛋白(bacterioferritin, Bfr)的编码基因atu2477和atu2771的功能。确定Bfr编码基因的开放阅读框。研究末端融合、血红素和个别关键氨基酸突变对Bfr功能和体外自组装的影响。探讨两种储铁蛋白的可能应用潜力。【方法】通过质粒将编码储铁蛋白的基因重新引入根癌农杆菌储铁蛋白缺失突变体中,回补储铁蛋白,验证回补的储铁蛋白编码基因是否能表达出具有储铁能力的储铁蛋白。用非变性凝胶电泳分离细胞粗提液中的蛋白质,铁特异性染色的方法鉴定电泳分离的蛋白质中是否有储铁蛋白。将不同的肽或蛋白质融合到储铁蛋白的末端,通过异源过量表达和纯化储铁蛋白的重组蛋白,用非变性凝胶电泳分析这些重组蛋白在体外的自组装。用血红素重构处理和氨基酸定点突变的方法研究血红素和个别关键氨基酸对Bfr功能和体外自组装的影响。【结果】非变性凝胶电泳和铁特异性染色结果显示,在根癌农杆菌的野生菌株、其相关突变体以及对应的回补菌株中,均仅检测到Bfr的表达,未检测到Dps的存在。当分别回补能编码161个和169个氨基酸Bfr的基因后,发现野生型菌株中的Bfr与回补编码161个氨基酸Bfr的回补菌株一样大。多肽和蛋白质的末端融合对Bfr的功能和自组装有一定影响,但不会使Bfr完全失去功能和自组装能力。结果还表明,血红素和预测可络合血红素铁的Met60的替换也只影响Bfr的功能和自组装,并未使Bfr功能完全丧失。【结论】根癌农杆菌主要通过Bfr存储铁元素。bfr基因的开放阅读框(open reading frame, ORF)以少见的UUG为起始密码子,编码产生包含161个氨基酸的蛋白质,而非169个氨基酸。根癌农杆菌的dps基因在本文的测定条件下均处于不表达状态。根癌农杆菌的Bfr和Dps蛋白均比较稳定,能够承受末端的多肽或蛋白质融合,不会使蛋白质的结构完全破坏,因此,经适当改造后具有开发应用的潜力。展开更多
Proteins, as the premier building blocks in nature, exhibit extraordinary ability in life activities during which process proteins mostly self-assemble into large complexes to exert prominent functions. Inspired by th...Proteins, as the premier building blocks in nature, exhibit extraordinary ability in life activities during which process proteins mostly self-assemble into large complexes to exert prominent functions. Inspired by this, recent chemical and biological stud- ies mainly focus on supramolecular self-assembly of proteins into high ordered architectures, especially the assembly suategy to unravel tile formation and function of protein nanostructures. In this review, we st, mmarize the progress made in the engi- neering of supramolecular protein architectures according to the strategies used to control the orient：ilion and the order of the assembly process. Furthermore, potential applications in biomedical areas of the supramolecular protein nanostructures will also be reviewed.展开更多
Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins.Rec...Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins.Recently,other types of molecular constraints,especially photoresponsive linkers and functional groups,have also found increased use in a wide variety of applications.Herein,we provide a concise review of using various forms of molecular strategies to constrain proteins,thereby stabilizing their native states,gaining insight into their folding mechanisms,and/or providing a handle to trigger a conformational process of interest with light.The applications discussed here cover a wide range of topics,ranging from delineating the details of the protein folding energy landscape to controlling protein assembly and function.展开更多
基金supported by the National Natural Science Foundation of China(21234004,21420102007,21574056,91527302)the Chang Jiang Scholars Program of China
文摘Proteins, as the premier building blocks in nature, exhibit extraordinary ability in life activities during which process proteins mostly self-assemble into large complexes to exert prominent functions. Inspired by this, recent chemical and biological stud- ies mainly focus on supramolecular self-assembly of proteins into high ordered architectures, especially the assembly suategy to unravel tile formation and function of protein nanostructures. In this review, we st, mmarize the progress made in the engi- neering of supramolecular protein architectures according to the strategies used to control the orient：ilion and the order of the assembly process. Furthermore, potential applications in biomedical areas of the supramolecular protein nanostructures will also be reviewed.
基金supported by the National Institutes of Health(GM-065978,AG-039253)
文摘Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins.Recently,other types of molecular constraints,especially photoresponsive linkers and functional groups,have also found increased use in a wide variety of applications.Herein,we provide a concise review of using various forms of molecular strategies to constrain proteins,thereby stabilizing their native states,gaining insight into their folding mechanisms,and/or providing a handle to trigger a conformational process of interest with light.The applications discussed here cover a wide range of topics,ranging from delineating the details of the protein folding energy landscape to controlling protein assembly and function.
