Interactions of fluorinated Surfactants with diphtheria toxin T-domain: Testing new media for studies of membrane proteins
Interactions of fluorinated Surfactants with diphtheria toxin T-domain: Testing new media for studies of membrane proteins
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DOI：
10.1529/biophysj.107.126235
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发表时间：
2008-06-01
影响因子：
3.4
通讯作者：
Ladokhin, Alexey S.
中科院分区：
文献类型：
--
作者：
Rodnin, Mykola V.;Posokhov, Yevgen O.;Ladokhin, Alexey S.
膜蛋白折叠和稳定性实验研究的主要困难是膜蛋白在脂双层外的聚集。为了解决这个问题，我们最近应用了氟化非洗涤剂表面活性剂作为化学伴侣。理想的伴侣表面活性剂将1）保持MP在溶液中; 2）最小程度地干扰MP的结构; 3）在膜插入期间从MP解离;和4）不分配到脂质双层中。在这里，我们比较了具有不同长度的半氟化（HFTAC）和完全氟化（FTAC）疏水链的表面活性剂如何与这种理想相比较。使用荧光相关光谱的染料标记的FTAC和HFTAC，我们证明，这两种类型的表面活性剂将结合脂囊泡。因此，与洗涤剂不同，氟化表面活性剂即使在远远超过其临界胶束浓度的浓度下也不会损害囊泡完整性。我们研究了表面活性剂与模型MP，DTT，使用各种光谱技术的相互作用。用荧光染料对DTT进行位点选择性标记表明，表面活性剂不会均匀地与DTT相互作用，而是集中在最疏水的斑块中。圆二色性测量表明，表面活性剂的存在不会改变DTT的结构。然而，表面活性剂的存在，特别是在临界胶束浓度以上（常规洗涤剂的一个特征），降低了热去折叠转变的协同性。DTT的去折叠焓对表面活性剂浓度的线性依赖性对于（H）FTAC的未来应用是令人鼓舞的，以确定其他MP的膜感受态构象的稳定性。观察到的供体标记的（H）FTAC和受体标记的DTT之间的福斯特共振能量转移的效率降低后，加入脂质囊泡表明，蛋白质脱落的表面活性剂层在其双层插入。我们讨论了氟化表面活性剂的优势，其他类型的增溶剂，特别强调其可能的应用在热力学测量。
The principal difficulty in experimental exploration of the folding and stability of membrane proteins (MPs) is their aggregation outside of the native environment of the lipid bilayer. To circumvent this problem, we recently applied fluorinated nondetergent surfactants that act as chemical chaperones. The ideal chaperone surfactant would 1), maintain the MP in solution; 2), minimally perturb the MP's structure; 3), dissociate from the MP during membrane insertion; and 4), not partition into the lipid bilayer. Here, we compare how surfactants with hemifluorinated (HFTAC) and completely fluorinated (FTAC) hydrophobic chains of different length compare to this ideal. Using fluorescence correlation spectroscopy of dye-labeled FTAC and HFTAC, we demonstrate that neither type of surfactant will bind lipid vesicles. Thus, unlike detergents, fluorinated surfactants do not compromise vesicle integrity even at concentrations far in excess of their critical micelle concentration. We examined the interaction of surfactants with a model MP, DTT, using a variety of spectroscopic techniques. Site-selective labeling of DTT with fluorescent dyes indicates that the surfactants do not interact with DTT uniformly, instead concentrating in the most hydrophobic patches. Circular dichroism measurements suggest that the presence of surfactants does not alter the structure of DTT. However, the cooperativity of the thermal unfolding transition is reduced by the presence of surfactants, especially above the critical micelle concentration (a feature of regular detergents, too). The linear dependence of DTT's enthalpy of unfolding on the surfactant concentration is encouraging for future application of (H) FTACs to determine the stability of the membrane-competent conformations of other MPs. The observed reduction in the efficiency of Forster resonance energy transfer between donor-labeled (H) FTACs and acceptor-labeled DTT upon addition of lipid vesicles indicates that the protein sheds the layer of surfactant during its bilayer insertion. We discuss the advantages of fluorinated surfactants over other types of solubilizing agents, with a specific emphasis on their possible applications in thermodynamic measurements.