NIH Director's Pioneer Award

我的长期目标是了解大分子拥挤对 通过获取蛋白质的原子级信息， 实际的生物条件。为此，我的团队开创了细胞内NMR。 我们已经展示了如何评估结构、量化动态和测量 在拥挤条件下的稳定性，在活的大肠杆菌细胞中发现， 这项强大的新技术。现在，我们想采取下一步。 在先锋奖的支持下，我们将专注于使用细胞内NMR， 真核细胞来研究神经退行性疾病中的两种关键蛋白质， 本质上无序的蛋白质，α-突触核蛋白和tau。这些蛋白质 优秀的候选人不仅因为他们的疾病相关性，而且 因为我们知道大分子拥挤对 无序蛋白质的特性。 我们对蛋白质结构和功能的理解在20世纪有了巨大的发展。 过去100年。我们已经从思考什么角色，如果有的话， 多肽在细胞中发挥作用，在原子水平上解开机制， 酶和蛋白质相互作用的分子基础， 了解人类疾病 我们积累的知识财富主要来自体外研究 在与生物学中发现的条件大不相同的条件下进行。为 例如，大多数蛋白质行为的生化检查都是在 浓度在？g至mg/mL范围，但细胞内部，其中大多数 蛋白质发挥作用，蛋白质浓度>300 mg/mL。 因此，我们的知识来自于在遥远的条件下获得的数据， 从生理学上讲，理论预测这些差异可能会导致 对生物物理参数的影响非常大。超越试管 通过使用NMR在活的真核细胞中进行真正的体内研究， 光谱学是蛋白质化学的新前沿。

My long term goal is to understand the effects of macromolecular crowding on biochemical processes by acquiring atomic-level information on proteins under actual biological conditions. To this end, my group has pioneered in-cell NMR. We have shown how to assess structure, quantify dynamics, and measure stability under the crowded conditions found in living Escherichia coli cells using this powerful new technique. Now, we want to take the next step. With support from a Pioneer Award, we will focus on using in-cell NMR in eukaryotic cells to study two key proteins in neurodegenerative diseases, the intrinsically disordered proteins, a-synuclein and tau. These proteins are excellent candidates not only because of their disease relevance, but also because we know that macromolecular crowding has extremely large effects on the properties of disordered proteins. Our understanding of protein structure and function has grown enormously in the last 100 years. We have progressed from pondering what role, if any, polypeptides play in the cell, to unraveling, at the atomic level, the mechanisms of enzymes and the molecular bases of protein-protein interactions vital to understanding human disease. Our accumulating wealth of knowledge has largely come from in vitro studies performed under conditions far different from those found in biology. For example, most biochemical examinations of protein behavior are performed at concentrations in the ?g-to-mg-per-mL range, but the insides of cells, where most proteins perform their work, have protein concentrations of >300 mg per mL. Thus, our knowledge comes from data acquired under conditions that are far from physiological relevant, and theory predicts these differences can have extremely large effects on biophysical parameters. Moving beyond the test tube by performing truly in vivo studies in living eukaryotic cells by using NMR spectroscopy is the next frontier in protein chemistry.