Deciphering the Mechanism of Transporters by Design and Experiments

描述(申请人提供)：转运蛋白是一类主要的膜蛋白，介导底物转位，与多种疾病相关的生理学有关，包括营养物质的异常细胞沉积，异常细胞死亡或增殖，以及药物的外流。因此，了解转运机制具有重要的理论意义，也是解决相关问题的基础，如分子水平上的疾病病因学和药物设计。尽管这些蛋白质很重要，但长期以来，由于这些蛋白质所在的双层环境的复杂性，实验上的困难一直阻碍着对运输机制的结构性理解。最近，结构研究的突破突显了共同转运机制的概念，揭示了功能不同的转运蛋白具有惊人的相似拓扑结构，由具有倒置对称性的双结构亚单位组成。在这里，我计划通过研究金属和糖的运输作为模型系统，利用实验、计算和结构生物物理的方法，对建立统一的运输机制进行基本的生物医学探索。在实施这项工作时，我的赞助人William DeGrado在蛋白质结构-功能-动力学关系、膜蛋白质的分子生物物理学和从头蛋白质设计等不同领域的领先研究将拓宽我在膜蛋白质结构和热力学折叠方面的专业知识。具体目标：将追求以下具体目标：目的1.为了检验对称对偶拓扑学在转运功能中的重要性，我将使用从头蛋白质设计方法在由对称设计的螺旋束组成的最简模型转运器中生成锌的转运功能。目的2.为了测试构象动力学在功能上的重要性，我将检验半乳糖转运蛋白vSGLT以及从头开始的锌转运蛋白多肽的活性是否反映了预期的构象变化的动力学行为。目的3.为了验证结构动力学在运输中重要性的推论，我将使用蛋白质设计和遗传筛选的方法来合成一种跨膜肽，通过与目标转运蛋白结合来变构锁定vSGLT或模型转运蛋白处于一种构象状态，并测试从头开始的变构结合肽对运输的抑制作用。再次强调该提案的创新之处：减少锌的易位与前列腺癌缺乏锌诱导的细胞凋亡有关，而特定的糖转运体在其他肿瘤细胞中上调，以满足增加的营养需求。因此，使用最低限度的锌转运蛋白和vSGLT作为模型系统来理解金属和糖的运输功能具有广泛的相关性。拟议项目的完成不仅将提供一个极好的培训机会，而且还将提出运输功能的概念性机械理论，这在治疗学的发展中具有潜在的重要意义。 与公共卫生相关：转运蛋白是驻留在生物膜中的蛋白质，控制各种分子进出细胞以及细胞细胞器的运动。由于维持化学平衡对细胞生存至关重要，了解转运蛋白的功能对于探索生理核心问题至关重要，例如转运蛋白功能是如何在分子水平上产生的，转运蛋白功能受损与癌症或心脏病等疾病的关系如何，以及药物设计。这一建议旨在提供对转运蛋白机制的概念性理解，以及选择性调节转运蛋白功能的新方法，实现这一点将具有重要的理论意义，也可能是治疗学发展的基础。

DESCRIPTION (provided by applicant): Transporters, a major class of membrane proteins, mediate substrate translocations that are implicated in a variety of disease-related physiology, including aberrant cellular deposition of nutrients, ab- normal cell death or proliferation, and efflux of drug. So, understanding the transport mechanism is theo- retically significant, as well as fundamental to addressing relevant questions, such as disease etiology at the molecular level and drug design. Despite the importance, efforts to gain structural understanding of transport mechanism have long been hampered by the experimental difficulty associated with the complex nature of the bilayer environment where these proteins reside. Very recently, an idea of common transport mechanism has been highlighted by the breakthroughs in structural investigations, which revealed that functionally diverse transporters share a surprisingly similar topology composed of dual structural subunits with inverted symmetry. Here, I plan to pursue basic biomedical quest on establishing a unified transport mechanism by investigating metal- and sugar-transportation as model systems, using methods of experimental, computational and struc- tural biophysics. In implementing the work, my expertise in membrane protein structure and thermodynamic folding will be broadened by the leading research of my sponsor, William DeGrado, in diverse fields of protein structure-function-dynamics relationship, molecular biophysics of membrane proteins and de novo protein design. Specific Aims: Following specific aims will be pursued: Aim 1. To test the importance of symmetric dual topo- logy in transport function, I will use de novo protein design approach to generate zinc transport function in a minimalist model transporter composed of a symmetrically designed helix bundle. Aim 2. To test the impor- tance of conformational dynamics in function, I will examine whether the activity by galactose transporter vSGLT, as well as de novo zinc transporter peptides, reflect the expected conformational change with respect to kinetic behaviors. Aim 3. To test the corollary of the importance of structural dynamics in transportation, I will use both protein design and genetic screening approaches to generate a transmembrane peptide that allosterically locks the vSGLT or model transporter in one conformational state by binding the target trans- porter, and test the transportation inhibition effect of the de novo allosteric binder peptide. Reemphasis of the proposal's innovation: Abated zinc translocation is related to the lack of zinc-induced apoptosis in prostate cancer, while specific sugar transporters are up regulated in other tumor cells to meet the increased demand for nutrient. So, understanding the metal- and sugar-transportation function using the minimalist zinc transporters and vSGLT as model systems is extensively relevant. Completion of the proposed project will not only provide an excellent training opportunity, but also present conceptual mechanistic theories for transport function, which is potentially important in therapeutics development. PUBLIC HEALTH RELEVANCE: Transporters are proteins that reside in the biological membrane and control the movement of variety of molecules in and out of cells, as well as cellular organelles. As maintaining the chemical balance is critical for cell survival, understanding how transporters function is central to exploring physiologically central questions, such as how transport function is generated at the molecular level, how impaired transport function is related to diseases, such as cancer or heart disease, and drug design. This proposal is aimed at providing the conceptual understanding for transporter mechanism, along with novel ways of selectively regulating the transport function, achieving of which will be theoretically significant, as well as potentially fundamental to therapeutics development.