New Approaches to Membrane Protein Structure

迄今为止测序的基因组中有相当大的比例被认为编码多聚体跨膜蛋白，这些蛋白催化多种基本细胞功能，特别是能量和信号转导。其中许多对人类疾病（如囊性纤维化、耐药性）和许多广泛开处方的药物(如。百忧解（Prozac）和奥美拉唑（proilosec）是针对膜转运蛋白的。尽管在过去的20年里，这类蛋白质的特性、纯化和修饰已经取得了进展，但只有少数蛋白质的研究达到了有助于理解其机制的水平。此外，许多膜蛋白需要构象灵活性才能发挥作用，因此获取动态结构信息势在必行。本申请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个氨基酸残基在机制上是不可替代的，并且应用新的位点导向生化和生物物理方法已经产生了一个螺旋包装模型，分辨率接近4埃单位。将进一步努力使用这些方法来完善和扩展该结构。此外，新开发的方法使用定向荧光共振能量转移和固态19F-NMR将被介绍。配体诱导的某些螺旋构象变化也可以被证明，这些研究将扩展到分子的其余部分，以描绘由配体结合引起的整体结构变化。底物结合位点位于螺旋IV和螺旋V之间的界面，特异性针对底物的半乳糖部分。合成了一种自旋标记的半乳糖苷，它与渗透酶具有高亲和力，并将用于进一步确定底物结合位点。结合但不易位的配体也被合成，以便研究在没有易位的情况下从膜的内表面和外表面结合。位点特异性烷基化结合质谱法将用于确定配体结合时His322（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.