Mechanisms of Membrane Translocation by Protein Toxins

描述(申请人提供)：该项目的目的是开发和测试结合蛋白质工程和同位素编辑傅里叶变换红外(FTIR)技术来分析蛋白质毒素引起的膜转位的分子机制的可行性。重点将放在白喉毒素(DT)上，这是一种包含受体结合(R)、跨膜(T)和催化(C)结构域的AB毒素。DT的T结构域促进了DT在胞浆转位过程中穿透内吞体膜。然而，其潜在的结构机制，即介导膜孔形成的T-结构域的构象转变，还不是很清楚。这一提议的总体假设是，DT的T结构域在孔形成和膜移位时发生主要构象变化，这将通过创新的生物物理方法来确定。选定的蛋白质亚基或片段将用13C稳定同位素标记，使用天然的和动态控制的多肽连接技术。偏振傅里叶变换红外光谱将被用来识别膜孔形成和蛋白质转运过程中DT的特定位置的构象和取向变化。在蛋白质膜插入过程中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。振动光谱分析亚基或链段选择性同位素标记蛋白质的结构是一种到目前为止还没有被利用的强大技术。这项技术的发展将有助于洞察蛋白质中定位的结构变化，这些变化是确定功能的基础。预计将在该项目内完成对具有稳定同位素的蛋白质中的整个亚基或大分子片段的标记和FTIR结构分析。将实现以下具体目标。具体目的1.制备亚基特异性稳定同位素标记的白喉毒素(DT)和分段标记的DT-T结构域，用于结构研究。将产生重组或半合成的蛋白质，其中的整个亚基或片段被稳定的同位素13C标记。首先，DT的13C标记的催化结构域将在大肠杆菌中表达，并将二硫键桥接到未标记的B链或T结构域上。其次，通过肽连接技术将产生几个T结构域的结构，其中定义的片段被选择性地标记为13C。这种方法将允许FTIR光谱分辨和识别促进膜孔形成和蛋白质移位的蛋白质的特定位置的构象和取向变化。特定目的2.确定白喉毒素及其T结构域的动态构象/取向变化，该结构域是细胞膜转位的基础。DT-T结构域在孔隙形成过程中经历主要构象/取向变化的假设将通过对亚单位和节段13C标记蛋白质的FTIR研究来验证。依赖时间的FTIR测量将揭示在膜插入和转位过程中蛋白质的二级结构和特定区域的取向的变化。13C标记的DT的C结构域和未标记的T结构域在膜转位过程中的构象变化将被识别。时间分辨荧光研究将揭示三级结构的动态变化和膜插入的动力学。

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.