NEW APPROACHES TO MEMBRANE PROTEIN STRUCTURES

描述：此应用程序的广泛、长期目标是 了解乳糖渗透酶的结构和作用机制 大肠杆菌作为二级运输蛋白的范例 将存储在电化学离子梯度中的自由能转化为功 以浓度梯度的形式。因为极少的残留物 在涉及广泛构象的机制中扮演关键角色 变化，似乎渗透酶周转涉及的相对简单 12个跨膜螺旋的刚体运动与化学耦合 组成分子的物质。因此，螺旋填料和螺旋填料的描绘 底物转运途径的定位对于 确定转运机制，乳糖渗透酶是唯一的 适合这个企业。拟议的具体目标是基于新的 开发的技术结合了蛋白质工程和各种 生物物理和生化方法能够提供 以接近原子级别的分辨率提供信息。12件商品的整体包装 渗透酶中的跨膜螺旋主要由以下因素决定 测量工程金属结合位点和定点定位之间的距离 氮氧化物自旋标记单半胱氨酸残基的电子顺磁研究 共振(EPR)。由金属-自旋标记距离得出的结论 然后，通过放置单个或成对的位置来确认和扩展测量 邻近螺旋中的半胱氨酸残基与定点准分子研究 荧光、自旋-自旋相互作用和肽键断裂 邻菲咯啉-铜。另一种方法将涉及配对的放置 His在相邻螺旋中的残基，并用EPR研究了Mn2+的结合。 最后，将荧光能量转移应用于渗透酶突变体。 含有单一的苯基萘磺酸盐标记的半胱氨酸残基和 背景中只有一个色氨酸残基，没有其他半胱氨酸和色氨酸残基。 底物转运途径将以高亲和力定位 自旋标记半乳糖苷和金属自旋标记距离测量 利用EPR。随后，人们将更精确地研究这条途径 研究自旋-自旋相互作用和荧光猝灭 自旋标记的半乳糖苷在含有单自旋-或的渗透酶构建物中 荧光标记的半胱氨酸残留物。次要活性转运蛋白有 涉及多种人类疾病，以及对某些疾病的抗药性 抗生素和某些药理制剂的作用机制。 这些转运蛋白中的许多似乎具有类似的二级结构，以 关于基因测序和水病特征，看起来很可能是 这些蛋白质的三级结构和机制一直是保守的。 因此，对细菌转运蛋白的研究更容易 操纵比它们的真核同行具有重要的相关性 高阶系统中的类似蛋白质，尤其是关于 开发新的结构-功能关系方法。

DESCRIPTION: The broad, long-term objectives of this application are to understand the structure and mechanism of the lactose permease of Escherichia coli as a paradigm for secondary transport proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. Since remarkably few residues play a critical role in a mechanism that involves widespread conformational changes, it seems likely that permease turnover involves relatively simple chemistry coupled to rigid body movements of the 12 transmembrane helices that compose the molecule. Thus, delineation of helix packing and localization of the substrate translocation pathway are essential for determining the transport mechanism, and the lactose permease is uniquely suited to this enterprise. the proposed specific aims are based on newly developed techniques that combine protein engineering and various biophysical and biochemical methods which are capable of providing information at near atomic-level resolution. The overall packing of the 12 transmembrane helices in the permease will be determined primarily by measuring distances between engineered metal binding sites and site-directed nitroxide spin labeled single-Cys residues using electron parmagnetic resonance (EPR). The conclusions derived from metal-spin label distance measurements will then be confirmed and extended by placing single or paired Cys residues in neighboring helices and studying site-directed excimer fluorescence, spin-spin interactions and peptide bond cleavage with o-phenanthroline-Cu. Another approach will involve the placement of paired His residues in neighboring helices and studying Mn2+ binding by EPR. Finally, fluorescence energy transfer will be applied to permease mutants containing a single anilinonaphthalene sulfonate-labeled Cys residue and a single Trp residue in a background devoid of other Cys and Trp residues. The substrate translocation pathway will be localized with high-affinity spin-labeled galactosides and metal-spin label distance measurements utilizing EPR. Subsequently, the pathway will be studied more precisely by studying spin-spin interactions and quenching of fluorescence by spin-labeled galactosides in permease constructs containing single spin- or fluorescent-labeled Cys residues. Secondary active transport proteins are involved in a variety of human diseases, as well as resistance to certain antibiotics and the mechanism of action of certain pharmacological agents. Many of these transporters appear to have similar secondary structures based on gene sequencing and hydropathy profiling and it seems likely that the tertiary structure and mechanism of these proteins have been conserved. Therefore, studies on bacterial transporters which are much easier to manipulate than their eukaryotic counterparts have important relevance to the analogous proteins in higher order systems, particularly with respect to the development of new approaches to structure-function relationships.