Membrane Protein Stability

描述(申请人提供)：膜蛋白是重要的药物靶标，在细胞中发挥重要作用，膜蛋白的错误折叠与人类疾病有关，如囊性纤维化。由于缺乏关于膜蛋白折叠和稳定性的数据，开发膜蛋白错误折叠疾病的治疗方法的能力受到很大限制。除了天然状态，我们还需要了解未折叠、部分折叠和错误折叠的膜蛋白的构象和稳定性。与几十年来一直在研究折叠的可溶性蛋白质相比，关于膜蛋白质折叠的热力学和力学信息的数据库很少。由于生物物理折叠研究可以提供序列-结构-功能关系的详细途径，而体内研究或晶体结构无法提供这些关系，因此有必要对膜蛋白进行更多的定量研究，以更好地了解它们的物理起源。在这项提案中，我们解决了关于膜蛋白折叠的信息缺乏的问题。我们将研究8种外膜蛋白：OmpX、OmpW、OmpA、PAgp、OmpT、OmpLa、Fad1和Omp85。我们的工作将使其折叠在脂质双层中的独特膜蛋白的数量翻一番。在第一个目标中，我们将建立体外条件，在这些条件下，它们将折叠成由天然脂肪提取物制备的膜。在第二个目标中，我们将使用动力学和热力学实验，使用SDS-PAGE、圆二色谱、荧光光谱和分析性超速离心法来确定折叠过程中涉及的步骤，并确定膜蛋白折叠倾向不同的原因。在最终目标中，我们解决了膜蛋白如何适应在其膜跨越区域的面向脂表面的可电离突变的引入。这些实验将深入了解遗传发生的可电离基团突变如何导致人类蛋白质功能障碍的机制。与公共卫生相关：对膜蛋白折叠的动态过程知之甚少，当膜蛋白错误折叠时，人类疾病就会发生。了解影响膜蛋白稳定性和膜蛋白折叠的因素，将有助于合理解释膜蛋白中发生的基因突变的影响。这些知识最终将有助于设计抗击疾病的治疗剂。

DESCRIPTION (provided by applicant): Membrane proteins are important pharmaceutical targets and play essential roles in cells, and misfolding of membrane proteins is associated with human diseases, such as cystic fibrosis. The ability to develop therapies for membrane protein misfolding diseases is substantially limited by the lack of data on membrane protein folding and stability. In addition to the native states, we need to understand the conformations and stabilities of unfolded, partially folded, and misfolded membrane proteins. Compared to soluble proteins, whose folding has been studied for decades, the database of thermodynamic and mechanistic information on membrane protein folding is minute. Since biophysical folding studies can provide detailed access to sequence-structure- function relationships that no in vivo studies or crystal structures can provide, there is a need for additional quantitative studies of membrane proteins to better understand their physical origins. In this proposal we address this lack of information on membrane protein folding. We will study 8 outer membrane proteins, OmpX, OmpW, OmpA, PagP, OmpT, OmpLa, FadL and Omp85. Our work will double the number of unique membrane proteins whose folding has been interrogated in lipid bilayers. In the first aim, we will establish in vitro conditions under which they fold into membranes prepared from native lipid extracts. In a second aim, we will use kinetic and thermodynamic experiments employing SDS-PAGE, circular dichroism, fluorescence spectroscopy and analytical ultracentrifugation to determine the steps involved in folding and to ascertain why membrane proteins differ in their folding propensities. In the final aim we address how membrane proteins accommodate the introduction of ionizable mutations on the lipid facing surfaces of their membrane spanning regions. These experiments will provide insight into the mechanisms of how genetically-occurring ionizable group mutations cause malfunctions in human proteins. PUBLIC HEALTH RELEVANCE: Little is known about the dynamical process of membrane protein folding, and human diseases occur when membrane proteins misfold. An understanding of the factors that influence membrane protein stability and membrane protein folding will find practical utility in rationalizing the effects of genetic mutations that occur in membrane proteins. This knowledge will ultimately be useful in the design of therapeutic agents to combat disease.