Structural Characterization of Protein and Protein Assemblies by Solid State NMR

活细胞内的结构通常是由一种蛋白质的许多副本组成的。就像由许多单独的砖块组成的砖墙一样，低聚物的大小和强度是单独的蛋白质无法达到的。例如，在细菌的生长和分裂过程中，在一种名为FtsZ的蛋白质的作用下，一个细胞被挤压成两个子细胞，许多拷贝的FtsZ组装成一个可以收缩的环。在另一个例子中，当细胞受到压力时，它们会通过停止细胞活动来做出反应。为了做到这一点，他们从许多拷贝的TIA-1中创建了一个蛋白质网状结构，通过将信使核糖核酸捕获在网状结构中来阻止蛋白质的合成。寡聚体结构可以对细胞内的事件非常敏感，远远超过单个蛋白质的敏感度，正如寡聚体血红蛋白的经典例子所表明的那样，低聚体血红蛋白对氧气的敏感度比单个血红蛋白高得多。虽然低聚结构至关重要，但人们对蛋白质分子如何聚集在一起形成更大的结构知之甚少。预计通过该项目获得的寡聚体结构的知识将加强我们对这些蛋白质在细胞中的生物学作用的理解。该项目将通过指导、教学和外联产生广泛影响。这位研究人员在指导女性科学方面有很好的记录，这些努力将继续下去。研究活动将通过面向初中生的推广活动来展示。尽管在过去的50年里，我们对蛋白质结构的知识有了很大的进步，但寡聚蛋白质尤其落后，因为它们很难用传统的结构方法进行研究。这个项目通过固体核磁共振技术的发展来追求低聚结构，固体核磁共振是一种很有前途的新方法，非常适合于确定低聚蛋白质的结构。在这些研究中，通过使用低温和通过将信号从电子自旋转移到核自旋，以一种称为动态核极化的方法来增强信号强度。本项目中实施的样品冷冻方案和多维探测方案的优化预计将改善低温线宽，并使确定结构的过程成为可能。虽然大多数研究将在体外样本中进行，但还开发了其他方法来阐明活细胞内的结构。细胞内的检测策略是使用选择性同位素浓缩和选择性地从标记在感兴趣蛋白质上的自由基转移极化的组合来选择性地增强感兴趣蛋白质的信号。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Structures inside living cells are often built from numerous copies of one protein. Like a brick wall made from many individual bricks, an oligomer can have size and strength unattainable by individual proteins. For example, during bacterial growth and division, a cell is pinched into two daughter cells by the action of a protein called FtsZ, many copies of which assemble into a ring that can constrict. In another example, when cells are stressed they respond by halting cellular activity. To do this they create a "mesh" of protein from many copies of TIA-1, which stalls protein synthesis by trapping mRNA in the "mesh". Oligomeric structures can be exquisitely sensitive to events in the cell, much more than an individual protein could be, as illustrated by the classic example of oligomeric hemoglobin which is much more sensitive to oxygen than a single hemoglobin protein. Although oligomeric structures are crucially important, little is known about how the protein molecules pack together to form the larger structures. It is expected that knowledge of the oligomeric structures gained through this project will enhance our understanding of the biological role of these proteins in the cell. This project will have broad impact through mentorship, teaching and outreach. The investigator has a strong record of mentoring women in science and these efforts will continue. Research activities will be showcased through outreach activities for middle and high school students.Although our knowledge of protein structure has advanced considerably over the past 50 years, oligomeric proteins specifically have lagged behind, because they are difficult to study using traditional structural methods. This project pursues oligomeric structures through technical developments in solid state NMR, a promising new method that is well suited for determining structures of oligomeric proteins. In these studies, the signal strength is enhanced through the use of low temperatures and by transfer of signal from electron spin to nuclear spin, in a method called Dynamic Nuclear Polarization. Optimization of sample freezing protocols, and multidimensional detection schemes pursued in this project are expected to improve the low temperature linewidths and enable the process of determining structures. Although most studies will be carried out with in vitro samples, additional methods are developed for elucidating structures within living cells. The strategy for detection within the cell is to selectively enhance signals for the oligomeric protein of interest using a combination of selective isotopic enrichment and selective transfer of polarization from radicals tagged onto the protein of interest.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.