Measurements of Protein Dynamics to Elucidate Protein and Enzyme Function.

测量蛋白质动力学以阐明蛋白质和酶的功能。

• 批准号: RGPIN-2016-06031
• 负责人: ONeil, Joe
• 金额: $ 2.26万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683956
• 关键词: Measurements; Protein; Dynamics; Elucidate; Enzyme

Proteins are molecular machines that carry out most of the work of a living cell. For example, they are responsible for synthesizing and repairing nearly all of the molecules that make up a cell. Proteins carry out their biological activities by changing their shapes in response to interactions with other molecules. This has been known for a long time but the detailed mechanisms of how proteins change their shapes have been difficult to determine. That's because measuring the details requires tracking the position of each atom in a protein on a timescale of billionths and millionths of a second. This field of study is called “protein dynamics” and it promises to reveal the deepest understanding of the mechanisms by which proteins function. Over the next 5 year cycle of my NSERC Discovery program, my students and I will apply the techniques of NMR spectroscopy, mass spectrometry, hydrogen exchange chemistry, X-ray diffraction and molecular dynamics simulations to measure the dynamics of three proteins over a wide range of timescales in order to connect the atomic dynamics to the emergent biological activity of each of the proteins. Advances in the field of protein dynamics will have a profound effect on our understanding of life and will have practical applications in biotechnology, industrial catalysis and drug design. The research will be conducted by graduate and undergraduate students who will be trained in the most advanced methods in the theory and practice of protein dynamics. ******The three proteins to be studied span the landscape of protein structural types including a highly flexible and disordered protein, a much more rigid protein and one that is intermediate between the two. The Transactivator of transcription from the Human Immunodeficiency Virus is highly flexible. We will measure its dynamics by NMR spectroscopy and hydrogen exchange chemistry and we will simulate its dynamics using computers. This work will contribute to a deeper understanding of how binding induces folding and the expression of function in this protein that is essential to viral replication. The glycerol facilitator is a relatively rigid integral membrane protein that permits the passive diffusion of glycerol across the bacterial inner membrane. We propose to measure protein dynamics on this protein in a membrane by measuring backbone amide hydrogen exchange rates with the use of mass spectrometry. These measurements will help delineate the role of dynamics in the transport of glycerol. The third protein we propose to investigate is the viral Ovarian Tumour domain deubiquitinase. It is an enzyme, a protein that catalyzes an important metabolic reaction involving ubiquitin. To probe the role of enzyme dynamics in the catalytic mechanism of this enzyme we will measure NMR relaxation rates over a range of temperatures in the presence and absence of binding partners to construct a comprehensive profile of the enzyme's dynamics. **

蛋白质是分子机器，可以进行大部分活细胞的工作。例如，它们负责合成和修复构成细胞的几乎所有分子。蛋白质通过改变与其他分子的相互作用来改变其形状，从而进行生物学活性。很长一段时间以来，这已经闻名了，但是很难确定蛋白质如何改变其形状的详细机制。这是因为测量细节需要在蛋白质中跟踪每个原子在十亿分和百万分之一秒的时间表上的位置。该研究领域称为“蛋白质动力学”，它有望揭示对蛋白质发挥作用机制的最深刻理解。在我的NSERC发现计划的接下来的5年周期中，我和我的学生将应用NMR光谱，质谱法，氢交换化学，X射线衍射和分子动力学模拟的技术，以测量三个蛋白质的动力学，以在各种时间表上连接到均具有蛋白质的动力学，以连接各种蛋白质的蛋白质活性。蛋白质动态领域的进步将对我们对生活的理解产生深远的影响，并在生物技术，工业催化和药物设计中具有实际应用。这项研究将由研究生和本科生进行，他们将接受蛋白质动力学理论和实践中最先进的方法的培训。 *****要研究的三种蛋白质跨越了蛋白质结构类型的景观，包括高度柔韧性和无序的蛋白质，一种更刚性的蛋白质和一种中间的蛋白质。来自人类免疫缺陷病毒转录的反式激活剂具有很高的灵活性。我们将通过NMR光谱和氢交换化学测量其动力学，并将使用计算机模拟其动力学。这项工作将有助于更深入地了解结合如何诱导对病毒复制至关重要的蛋白质功能表达。甘油促进剂是一种相对刚性的整体膜蛋白，可允许甘油在细菌内膜中被动扩散。我们建议通过使用质谱法测量骨干酰胺氢汇率来测量膜上该蛋白质的蛋白质动力学。这些测量将有助于描述动力学在甘油运输中的作用。我们建议研究的第三种蛋白质是病毒卵巢肿瘤域去泛素酶。它是一种酶，一种蛋白质，可催化涉及泛素的重要代谢反应。为了探测酶动力学在该酶的催化机制中的作用，我们将在存在和不存在结合伴侣的情况下测量一系列温度的NMR弛豫率，以构建酶动力学的全面概况。 **