Ion Gradients and Energy Coupling in Bacteria

描述(由申请人提供)：这项工作的长期目标是在基础水平上了解膜转运蛋白的结构和其功能的机制特征之间的关系。我们正在研究的模型蛋白UhpT负责大肠杆菌对己糖磷酸的吸收和运输，也代表了其在主要促进器超家族中的亲属，该家族是膜运输系统的最大集合之一。这种相关系统(I)促进糖在所有哺乳动物细胞膜上的移动，包括那些参与胰岛素反应的细胞膜；(Ii)被招募来组织中枢神经系统中神经递质的分泌；以及(Iii)在病原菌的耐药性中发挥中心作用。因此，了解我们模型(UhpT)中的转运机制也将有助于我们理解大量与人类健康抗病相关的系统。我们计划进行四条线的实验研究，以了解我们的模型蛋白质UhpT中的结构/功能关系。(1)利用特殊构建的双半胱氨酸突变体，通过二硫键捕获和其他交联法来探索螺旋-螺旋邻近的一般特征。(2)在合作中，半胱氨酸取代突变将被用于植入支持电子顺磁共振(EPR)的探针，以精确判断底物结合过程中螺旋-螺旋距离的变化。(3)继续的研究将利用定点和选择定向突变来揭示排列在易位途径上的残基的性质。这应该说明底物专一性和选择性的基础原理。(4)最后，在与美国国立卫生研究院同事的持续合作中，针对两种UhpT相关蛋白质(GIpT和UHPC)的2D和3D结晶学的研究应该允许直接和具体的证据支持通过更间接的方法得出的结论。总之，这种遗传、生化和生物物理方法的结合应该揭示膜运输现象背后的结构相关性。

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand, at a fundamental level, the relationship between the structure of a membrane transport protein and the mechanistic features of its function. The model protein we are studying, known as UhpT, is responsible for the uptake and transport of hexose phosphates by Escherchia coli and serves also to represent its relatives in the Major Facilitator Superfamily, one of the largest collections of membrane transport systems. Such related systems (i) facilitate movement of sugar across all mammalian cell membranes, including those involved in the response to insulin; (ii) are recruited to organize the secretion of neurotransmitters in the central nervous system; and (iii) act as central players in the drug resistance of pathogenic bacteria. For these reasons, understanding the mechanism of transport in our model (UhpT) will also help us understand a large number of systems relevant to human health antidisease. Four lines of experimental study are planned to understand structure/function relationships in our model protein, UhpT. (1) By using specially constructed double-cysteine variants, the general features of helix-helix proximity will be probed by disulfide trapping and other cross-linking protocols. (2) In a collaborative effort, cysteine-substitution mutagenesis will be used to implant probes supporting electron paramagnetic spectroscopy (EPR) to precisely judge changes in helix-helix distance during substrate binding. (3) Continuing studies will exploit both site- and selection-directed mutagenesis to reveal the properties of residues lining the translocation pathway. This should illuminate principles that underlie substrate specificity and selectivity. (4) Finally, in an ongoing collaboration with colleagues at the National Institutes of Health, studies aimed at both 2D and 3D crystallography of two UhpT-related proteins (GIpT and UhpC) should allow direct and concrete evidence supporting conclusions drawn by the more indirect approaches. Together, this combination of genetic, biochemical and biophysical approaches should reveal the structural correlates underlying the phenomenon of membrane transport.