Mechanisms of Membrane Translocation by Protein Toxins

项目描述（由申请人提供）：本项目旨在开发并测试蛋白质工程和同位素编辑傅立叶变换红外（FTIR）技术相结合的可行性，以分析蛋白质毒素引起的膜易位的分子机制。重点将放在白喉毒素（DT）上，这是一种含有受体结合(R)、跨膜(T)和催化(C)结构域的AB毒素。胞质溶胶易位过程中DT通过胞内体膜是由其t结构域促进的。然而，潜在的结构机制，即介导膜孔形成的t结构域的构象转变，尚未得到很好的理解。该提案的总体假设是，DT的t结构域在孔隙形成和膜易位时发生了主要的构象变化，这将通过创新的生物物理方法来识别。选定的蛋白质亚基或片段将使用天然和动力学控制的肽连接技术用13C稳定同位素进行标记。极化FTIR光谱将用于识别在膜孔形成和蛋白质易位过程中DT的位点特异性构象和取向变化。在膜插入过程中，蛋白质的实时结构变化将通过停流荧光和时间分辨FTIR光谱进行监测。利用振动光谱技术对亚基或片段选择性同位素标记的蛋白质进行结构分析是一种强大的技术，但迄今尚未得到应用。这项技术的发展将有助于深入了解蛋白质中定位的结构变化，这些变化是定义功能的基础。预计该项目将完成稳定同位素对蛋白质整个亚基或大片段的标记和FTIR结构分析。将实现以下具体目标。具体目标生产亚单位特异性稳定同位素标记白喉毒素（DT）和区段标记DT的t结构域进行结构研究。重组蛋白或半合成蛋白可以用稳定同位素13C标记整个亚基或片段。首先，统一的13c标记的DT催化结构域将在大肠杆菌中表达，二硫化物桥接到未标记的b链或t结构域。其次，通过肽连接技术将产生若干t结构域结构体，其中已定义的片段被选择性地标记为13c。这种方法将允许FTIR光谱分辨率和鉴定蛋白质中促进膜孔形成和蛋白质易位的位点特异性构象和取向变化。具体目标2。确定白喉毒素及其T结构域在膜易位基础上的动态构象/取向变化。DT - t结构域在孔隙形成过程中经历了主要构象/取向变化的假设将通过对亚基和片段13c标记的蛋白质进行FTIR研究来验证。随时间变化的FTIR测量将揭示在膜插入和易位过程中蛋白质二级结构和特定区域方向的变化。在膜易位过程中，13C标记的DT的c结构域和未标记的t结构域的构象变化将被确定。时间分辨荧光研究将揭示三级结构的动态变化和膜插入动力学。

DESCRIPTION (provided by applicant): The aim of this project is to develop and test for feasibility combined protein engineering and isotope edited Fourier transform infrared (FTIR) techniques to analyze the molecular mechanisms of membrane translocation by protein toxins. The focus will be on diphtheria toxin (DT), an AB toxin that contains the receptor binding (R), transmembrane (T), and catalytic (C) domains. The crossing of the endosome membrane by DT during cytosol translocation is facilitated by its T-domain. However, the underlying structural mechanisms, i.e. the conformational transitions in the T-domain that mediate membrane pore formation, are not well understood. The overall hypothesis of this proposal is that major conformational changes occur in the T-domain of DT upon pore formation and membrane translocation, which will be identified by innovative biophysical approaches. Selected subunits or segments of the protein will be labeled with the 13C stable isotope using native and kinetically controlled peptide ligation techniques. Polarized FTIR spectroscopy will be used to identify site- specific conformational and orientational changes in DT during membrane pore formation and protein translocation. Real-time structural changes during membrane insertion of the protein will be monitored by stopped-flow fluorescence and time-resolved FTIR spectroscopy. Structural analysis of subunit- or segment- selective isotope labeled proteins by vibrational spectroscopy is a powerful technique that has not been utilized thus far. Development of this technique will help gain insight in site-resolved structural changes in proteins that underlie defined functions. It is anticipated that labeling of whole subunits or larg segments within a protein with stable isotopes and structural analysis by FTIR will be accomplished within this project. The following Specific Aims will be pursued. Specific Aim 1. Produce subunit-specific stable isotope-labeled diphtheria toxin (DT) and segmentally labeled T-domain of DT for structural studies. Recombinant or semisynthetic proteins will be produced in which a whole subunit or a segment is labeled with the stable isotope 13C. First, the uniformly 13C-labeled catalytic domain of DT will be expressed in E. coli and disulfide bridged to the unlabeled B-chain or the T-domain. Second, several constructs of the T-domain in which defined segments are selectively 13C-labeled will be produced by peptide ligation techniques. This approach will allow FTIR spectral resolution and identification of site-specific conformational and orientational changes in proteins that facilitate membrane pore formation and protein translocation. Specific Aim 2. Identify the dynamic conformational/orientational changes in diphtheria toxin and its T domain that underlie membrane translocation. The hypothesis that DT T-domain undergoes major conformational/orientational changes during pore formation will be tested by FTIR studies on the subunit- and segmental- 13C-labeled protein. Time- dependent FTIR measurements will reveal changes in the secondary structure and the orientation of defined regions of the protein during membrane insertion and translocation. Conformational changes in both the 13C- labeled C-domain and unlabeled T-domains of DT during membrane translocation will be identified. Time- resolved fluorescence studies will reveal dynamic changes in the tertiary structure and the kinetics of membrane insertion. PUBLIC HEALTH RELEVANCE: Protein toxins need to cross one or more cellular membranes to enter the cell and damage or kill it. The mechanisms of membrane translocation are not well understood and require further studies. The aim of this project is to develop and test for feasibility advanced protein engineering and biophysical approaches to analyze the molecular mechanisms of membrane translocation by protein toxins, such as diphtheria toxin. Better understanding of the molecular mechanisms of protein toxins will facilitate development of novel anti- toxin therapies.