New Approaches to Membrane Protein Structure

到目前为止，测序的基因组中有很大比例被认为编码多位跨膜蛋白，这些蛋白催化多种基本的细胞功能，特别是能量和信号转导。许多药物对人类疾病(例如囊性纤维化、耐药性)和许多广泛开出的药物(例如，百忧解和Prilosec)针对的是膜运输蛋白。虽然在过去的20年里，对这类蛋白质的表征、纯化和修饰已经取得了进展，但在有助于理解其机制的水平上进行的研究寥寥无几。此外，许多膜蛋白需要构象灵活性才能发挥作用，这使得获得动态结构信息势在必行。这项应用的目的是继续利用大肠杆菌的乳糖渗透酶作为跨膜蛋白结构/功能研究的范例。只有6个氨基酸残基在机理上是不可替代的，新的定点定向生化和生物物理方法的应用产生了一个螺旋堆积模型，分辨率约为4埃单位。将利用这些方法进一步努力改进和扩展结构。此外，还将介绍使用定点荧光共振能量转移和固体~(19)F-核磁共振的新开发方法。还可以证明配体诱导的某些螺旋的构象变化，这些研究将扩展到分子的其余部分，以描绘由于配体结合而导致的整体结构变化。底物结合位置位于螺旋IV和V之间的界面，特异性针对底物的半乳糖部分。合成了一种高亲和力的自旋标记半乳糖苷，并将其用于进一步确定底物结合部位。也正在合成结合但不转位的配体，以便在没有转位的情况下研究膜内和外表面的结合。特定部位的烷基化结合质谱学将被用来确定His322(螺旋X)与配体结合时质子化的变化。

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.