New Approaches to Membrane Protein Structure

到目前为止，被测序的基因组中有很大比例被认为编码多位跨膜蛋白，其催化多种基本的细胞功能，特别是能量和信号转导。 许多是重要的关于人类疾病（如囊性纤维化，耐药性），和许多广泛的处方药（如。Prozac和Prilosec）靶向膜转运蛋白。 虽然在过去的20年中取得了进展，导致这类蛋白质的表征，纯化和修饰，只有少数已经在了解机制有用的水平进行了研究。 此外，许多膜蛋白需要构象的灵活性，以发挥作用，使其必须获得动态结构信息。本申请的目的是继续利用大肠杆菌的乳糖通透酶作为跨膜蛋白的结构/功能研究的范例。 只有6个氨基酸残基是不可替代的机制，和应用新的定点生物化学和生物物理方法已经产生了一个螺旋包装模型的分辨率接近4埃单位。 将进一步努力使用这些方法改进和扩展结构。此外，还将介绍新开发的使用定点荧光共振能量转移和固态19 F-NMR的方法。 配体诱导的构象变化，在某些螺旋也可以证明，这些研究将扩展到分子的其余部分，以描绘整体结构变化，配体结合的结果。底物结合位点位于螺旋IV和V之间的界面处，特异性针对底物的半乳糖基部分。 自旋标记的半乳糖苷，结合的通透酶具有高亲和力已被合成，并将用于进一步定义的底物结合位点。 也正在合成结合但不移位的配体，以研究在不存在移位的情况下从膜的内表面和外表面的结合。 位点特异性烷基化结合质谱法将用于测定配体结合后His 322（螺旋X）质子化的变化。

A highly significant percentage of the genomes sequenced thus far are thought to encode polytopic transmembrane proteins which catalyze a multitude of essential cellular functions, energy and signal transduction in particular. Many are important with regard to human disease (e.g. cystic fibrosis, drug resistance), and many widely prescribed drugs (eg. Prozac and Prilosec) are targeted to membrane transport proteins. Although progress over the last 20 years has led to the characterization, purification and modification of this class of proteins, only a few have been studied at a level useful for understanding mechanism. Furthermore, many membrane proteins require conformational flexibility in order to function, making it imperative to obtain dynamic structural information. The objectives of this application are to continue to utilize the lactose permease of Escherichia coli as a paradigm for structure/function studies on transmembrane proteins. Only 6 amino acid residues are irreplaceable with respect to mechanism, and application of novel site-directed biochemical and biophysical approaches has yielded a helix packing model to a resolution approximating 4 Angstrom units. Further efforts will be made to refine and extend the structure using these methods. In addition, newly developed approaches using site-directed fluorescence resonance energy transfer and solid-state 19F-NMR will be introduced. Ligand-induced conformational changes in certain helices can also be demonstrated, and these studies will be extended to the remainder of the molecule in order to delineate overall structural changes that result from ligand binding. The substrate binding site is located at the interface between helices IV and V, and specificity is directed towards the galactosyl moiety of the substrate. A spin-labeled galactoside that binds to the permease with high affinity has been synthesized and will be used to further define the substrate binding site. Ligands that bind but are not translocated are also being synthesized in order to study binding from the inner and outer surface of the membrane in the absence of translocation. Site-specific alkylation combined with mass spectrometry will be used to determine changes in the protonation of His322 (helix X) upon ligand binding.