EFFECT OF HEMIFLUORINATED SURFACTANTS ON MEMBRANE INSERTION/FOLDING OF DIPHTHER

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。白喉毒素t结构域（DTT）的功能是将毒素的催化结构域转移到脂质双分子层上，以响应内体的酸化，这是许多细菌毒素（包括潜在的生物武器肉毒杆菌）共有的一种进入模式。尽管DTT的膜相互作用的表征取得了进展，但其作用的分子机制和DTT在其功能膜插入形式下的结构仍不清楚。半氟化合物，如HF-TAC，是一类新型的非洗涤剂表面活性剂，旨在改善膜蛋白的增溶作用，用于功能和结构研究。由于DTT（取决于pH值）可以以插入型和水溶性形式存在，因此它为研究这些表面活性剂对膜蛋白插入/折叠途径的一般影响提供了一个方便的模型。这项拨款的目的是利用定点荧光标记和其他光谱方法确定表面活性剂与DTT相互作用的模式。具体目的是：(1)确定HF-TAC对DTT聚集状态的影响；(2)确定HF-TAC对DTT膜结合自由能的影响；(3)确定HF-TAC对dtt诱导的膜破坏的影响；(4)确定HF-TAC对脂质囊泡完整性的直接影响。本研究将为后续膜环境下DTT的高分辨率结构研究奠定基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The function of diphtheria toxin T-domain (DTT) is to translocate the catalytic domain of the toxin across the lipid bilayer in response to acidification of the endosome, a mode of entry shared by a number of bacterial toxins, including the potential bioweapon botulinum. Despite the progress in characterization of membrane interactions of DTT, the molecular mechanism of its action and the structure of DTT in its functional membrane-inserted form remain unknown. Hemifluorinated compounds, such as HF-TAC, are a novel class of non-detergent surfactants designed to amend solubilization of membrane proteins for functional and structural studies. Because DTT, depending on pH, can be found in both an insertion-competent and water-soluble form, it presents a convenient model for studying the general effects of these surfactants on the membrane protein insertion/folding pathway. The objective of this grant is to determine the mode of interaction of surfactants with DTT using site-directed fluorescence labeling and other spectroscopic approaches. The specific aims are: (1) determine the effect of HF-TAC on the aggregation state of DTT; (2) determine the effect of HF-TAC on the free energy of membrane association of DTT; (3) determine the effect of HF-TAC on DTT-induced membrane disruption; (4) determine the direct effect of HF-TAC on the integrity of the lipid vesicles. This study will lay the groundwork for subsequent high resolution structural studies of DTT in membrane environment.