Protein Activity and Oligomer Stability in Cell Membrane

智力优势：细胞膜的选择性渗透性是我们所知的所有生命形式所必需的，是由膜蛋白赋予的。 大约80%的膜蛋白具有已知的结构，存在的低聚物结晶时，表明大部分的蛋白质作为低聚物在细胞膜中的功能。 然而，关于蛋白质如何在细胞膜中寡聚化以及寡聚化如何决定蛋白质功能的问题仍然存在。 对这些问题的深入理解将对许多研究领域产生巨大的影响，包括细胞膜的生物发生，稳态的建立，信号转导，跨细胞膜的物质运输和膜中蛋白质功能的调节。 作为对寡聚体形成偏好的基本理解的一步，该项目旨在测试以下假设：膜蛋白的寡聚体稳定性被优化以支持功能，并且稳定性的显著增加将降低活性。 大肠杆菌蛋白AcrB将用作模型。 将通过产生和表征具有降低或增加的寡聚体稳定性的AcrB突变体来测试该假设。 将使用一系列生物化学和生物物理方法，包括定点和随机诱变、蛋白质提取和纯化、蛋白质活性测定、二硫键捕获、凝胶电泳、圆二色性和荧光光谱。 本研究的结果将揭示四级结构稳定性对AcrB功能的影响。 这项研究预计将产生一些有关多结构域和多跨度螺旋膜蛋白的寡聚体稳定性与其体内活性之间相关性的首批数据。 更广泛的影响：这项工作将涉及从高中到攻读博士学位的研究生候选人的广泛的学生培训。度 两名研究生将接受培训，以解决膜蛋白结构，功能，折叠和相互作用研究领域的科学问题。 此外，两至三名本科生和高中生将参加拟议的研究，通过他们将获得在现代生物化学进行研究的实践经验。 将特别关注代表性不足和少数民族本科生的招聘和教育。 PI将为学生提供在多学科和多元文化环境中工作的机会，为日益国际化和全球化的工作环境做好准备。 拟议研究的发现将通过在同行评审的期刊上发表文章和在研究会议上发言，向广大科学界传播。

Intellectual Merit: The selective permeability of cell membranes, which is essential for all life forms that we know, is conferred by membrane proteins. Approximately 80% of membrane proteins with known structures exist as oligomers when crystallized, indicating a large portion of proteins function as oligomers in cell membranes. However, questions remain about how proteins oligomerize in the cell membrane and how oligomerization determines protein functions. A thorough understanding of these questions will have an immense impact on many research areas including the biogenesis of cell membranes, establishment of homeostasis, signal transduction, material transport across cell membranes and regulation of protein functions in membranes. As a step toward a fundamental understanding of the preference for oligomer fomation, this project is designed to test the hypothesis that oligomer stabilities of membrane proteins are optimized to support function and a significant increase of stability will decrease activity. Escherichia coli protein AcrB will be used as a model. The hypothesis will be tested through creation and characterization of AcrB mutants with decreased or increased oligomer stability. An array of biochemical and biophysical methods will be used, including site-directed and random mutagenesis, protein extraction and purification, a protein activity assay, disulfide trapping, gel electrophoresis, circular dichroism and fluorescence spectroscopy. Outcomes from this research will reveal the influence of quaternary structure stability on AcrB function. This study is expected to generate some of the first data pertaining to the correlation between oligomer stability of multi-domain and multi-span helical membrane proteins and their in vivo activity. Broader Impacts: This work will involve a wide range of student training from high school to graduate candidates pursing Ph.D. degrees. Two graduate students will be trained to tackle scientific questions in the research area of membrane protein structure, function, folding, and interaction. In addition, two to three undergraduate and high school students will participate in the proposed research, through which they will gain hands-on experience in undertaking research in modern biochemistry. Special focus will be given to the recruitment and education of underrepresented and minority undergraduate students. The PI will provide an opportunity for students to work in a multidisciplinary and multicultural environment, preparing them for the increasingly international and global working environment. The discoveries resulting from the proposed research will be disseminated to scientific communities at large, through publications in peer-reviewed journals and presentations at research conferences.