Macromolecular Crowding and Protein Stability In Vitro and in Cells

总体目标是了解大分子拥挤对蛋白质化学的影响。重点是平衡蛋白的稳定性。实验将在体外和细胞中进行。核磁共振波谱将用于评估拥挤引起的稳定性变化和蛋白质-拥挤相互作用。两个未解决的问题将得到解决。问题1：拥挤与测试蛋白之间的哪些相互作用决定了拥挤对稳定性的影响？拥挤效应可分为体积排斥和化学相互作用两部分，它们的相对重要性是拥挤中最关键的未解决问题。问题2：哪些拥挤因子提供了最相关的生理信息？合成聚合物，如聚乙烯基吡罗烷酮（PVP）、菲科尔（Ficoll）、聚乙二醇（PEG）和葡聚糖（Dextran），经常用于拥挤研究，但它们的生物学相关性必须进行评估。为了解决这些问题，我们将测试三个假设。第一个假设涉及合成聚合物作为拥挤剂。现在我们知道，蛋白质的稳定性可以在充满PVP的溶液中进行评估。该研究项目将探索其他常用的聚合物：Ficoll、Dextran和PEG。假设PVP、Ficoll和右旋糖酐主要通过体积排斥起作用，化学相互作用不太重要。这一结果对稳定化学和制药工业中重要的蛋白质具有启示意义。第二个假设涉及到蛋白质聚集。假设是不稳定的非特异性化学相互作用之间的蛋白质拥挤和测试蛋白质被低估了。预测是蛋白质的拥挤会导致不稳定。这些结果对理解细胞过程具有启示意义。第三种假设侧重于最终的生物学相关性。假设是在大肠杆菌的细胞质中，稳定的排除体积效应被非特异性化学相互作用所抵消。更广泛的影响许多增加科学多样性的努力都是为了识别和吸引个别高中生。目前的奖项将用于激励来自学生结构多样化和大学出勤率低的学校的科学教师。计划是每年夏天邀请一位这样的北卡罗莱纳高中科学老师到实验室呆七周。位于北卡罗来纳大学的全国大学咨询服务团的任务是将新近毕业的大学毕业生安置在大学出勤率较低的高中，以减少进入大学的障碍。军团通过访问学校直接与学校互动。此资源将用于确定教师，谁将被鼓励申请。老师将在研究小组内参与这个项目。首席研究员每周至少与教师会面一次，制定课程计划，向他或她的学生介绍研究。PI还将与生物技术和制药行业的专业人士举行会议。例如，三角研究园区当地生物技术公司的领导可以解决药物从实验室到患者的路径问题，北卡罗来纳大学技术发展办公室的代表可以解决知识产权问题。

Intellectual MeritThe overall goal is to understand the effects of macromolecular crowding on protein chemistry. The focus is on equilibrium protein stability. Experiments will be performed both in vitro and in cells. Nuclear resonance spectroscopy will be used to assess both crowding induced changes in stability and protein-crowder interactions. Two unresolved questions will be addressed. Question 1: Which interactions between the crowder and the test protein determine the effects of crowding on stability? Crowding effects can be divided into two parts: volume exclusion and chemical interactions and their relative importance is the most critical unresolved question in crowding. Question 2: Which crowding agents provide the most physiologically relevant information? Synthetic polymers, like Poly Vinyl Pyrrolidone (PVP), Ficoll, Poly Ethylene Glycol (PEG), and Dextran, are often used in crowding studies, but their biological relevance has to be assessed. In an effort to address these questions three hypotheses will be tested. The first hypothesis involves synthetic polymers as crowding agents. It is now known that protein stability can be assessed in solutions crowded with PVP. The research project will explore other commonly used crowders: Ficoll, Dextran, and PEG. The hypothesis is that PVP, Ficoll, and Dextran work mostly by volume exclusion, and chemical interactions will be of minor importance. The results have implications for stabilizing proteins important to the chemical and pharmaceutical industries.The second hypothesis involves protein crowders. The hypothesis is that destabilizing non-specific chemical interactions between protein crowders and the test protein have been underestimated. The prediction is that crowding by proteins can lead to destabilization. The results have implications for understanding cellular processes.The third hypothesis focuses on the ultimate biological relevance. The hypothesis is that in the cytoplasm of Escherichia coli, the stabilizing excluded volume effect is offset by non-specific chemical interactions.Broader ImpactsMany efforts to increase scientific diversity are designed to identify and attract individual high school students. The present award will be used to leverage these efforts by invigorating science teachers from schools with diverse student bodies and low college attendance. The plan is to bring one such North Carolina high school science teacher to the lab for seven weeks each summer.The mission of the National College Advising Corps, located at UNC, is to place recent college graduates in high schools with low college attendance in an attempt to reduce barriers to college access. The Corps interacts directly with the schools by visiting them. This resource will be used to identify the teachers, who would be encouraged to apply. The teacher will work within the research group on the project. The Principal Investigator will meet at least once a week with the teacher to develop a lesson plan to introduce his or her students to research. The PI will also set up meetings with professionals from the biotech and pharmaceutical industries. As an example, leaders from local biotech firms in Research Triangle Park could address the path a drug takes from the lab to the patient, and a representative from the UNC Office of Technology Development could address intellectual property issues.