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. **

蛋白质是执行活细胞大部分工作的分子机器。例如，它们负责合成和修复几乎所有构成细胞的分子。蛋白质通过响应与其他分子的相互作用而改变其形状来发挥其生物活性。人们很早就知道这一点，但蛋白质如何改变其形状的详细机制一直难以确定。 这是因为测量细节需要在十亿分之一秒和百万分之一秒的时间尺度上跟踪蛋白质中每个原子的位置。 这一研究领域被称为“蛋白质动力学”，它有望揭示对蛋白质功能机制的最深入理解。在我的 NSERC 发现计划的下一个 5 年周期中，我和我的学生将应用核磁共振波谱、质谱、氢交换化学、X 射线衍射和分子动力学模拟技术来测量三种蛋白质在广泛时间尺度上的动力学，以便将原子动力学与每种蛋白质的新兴生物活性联系起来。蛋白质动力学领域的进步将对我们对生命的理解产生深远的影响，并将在生物技术、工业催化和药物设计中产生实际应用。该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最先进方法的培训。 ******要研究的三种蛋白质跨越了蛋白质结构类型的范围，包括一种高度灵活且无序的蛋白质、一种更加刚性的蛋白质以及一种介于两者之间的蛋白质。人类免疫缺陷病毒转录的反式激活因子高度灵活。我们将通过核磁共振波谱和氢交换化学来测量其动力学，并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折叠以及该蛋白质的功能表达，这对于病毒复制至关重要。甘油促进剂是一种相对刚性的整合膜蛋白，允许甘油被动扩散穿过细菌内膜。我们建议通过使用质谱测量主链酰胺氢交换率来测量膜中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结构域去泛素酶。它是一种酶，一种蛋白质，可催化涉及泛素的重要代谢反应。为了探究酶动力学在该酶催化机制中的作用，我们将在存在和不存在结合伴侣的情况下测量一定温度范围内的 NMR 弛豫率，以构建酶动力学的全面概况。 **