Single-molecule mass photometry to probe the competition between protein aggregation and native folding

单分子质量光度法探测蛋白质聚集和天然折叠之间的竞争

• 批准号: RTI-2020-00301
• 负责人: Woodside, Michael
• 金额: $ 10.51万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 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=693582
• 关键词: Single; molecule; mass; photometry; probe

Proteins fold into complex structures that are required for correct function. These structures involve frustrated' interactions that can't always be satisfied, allowing alternative structures that cause dysfunction to form. Indeed, many diseases are linked to aggregates of such misfolded proteins. Understanding how aggregation competes with native folding will yield key insights into how folding goes wrong and improved principles for designing new proteins. We will probe aggregation using interferometric light scattering to measure the mass of single proteins in solution. Because this method is sensitive to single molecules, it can monitor the earliest steps of aggregationsmall oligomerswithin a heterogeneous mixture covering all stages of the aggregation cascade; because it is label-free, aggregation can be probed without any bias from the fluorescent dyes normally used for detection. We will use single-molecule (SM) mass photometry to distinguish oligomers of different size (dimer, trimer,) and catalog their formation/growth rates, obtaining for the first time an unbiased, quantitative measure of each state in the aggregation cascade for comparison to physical models. We will probe 2 specific aims:***1. How proteins switch from native to aggregated when conditions change. We will focus on the tau protein as an example: it normally helps stabilize microtubules in neurons, but switches to aggregate in Alzheimer's disease and other dementias. We will compare the formation/growth rates of oligomers under normal and diseased conditions, fitting to microscopic models to deduce the mechanism driving the change in behaviour.***2. How evolution shapes the competition between native folding and aggregation. Evolution is thought to select for smooth energy landscapes that allow rapid formation of minimally frustrated structures. To study this question, we will (A) compare aggregation of ancestral proteins from different evolutionary periods, focusing on PrP, a protein that aggregates to cause disease and whose sequence we have reconstructed back to ~350 million years ago; and (B) measure aggregation of proteins designed computationally, which have not undergone evolutionary selection and are often prone to aggregation even though de novo design is increasingly reliable. In each case, we will compare the formation/growth rate of oligomers measured by SM mass photometry to properties of the native folding like stability, barrier height, and landscape roughness that we are measure using SM force spectroscopy, to identify the physical changes in the folding related to differences in aggregation-propensity.***Such studies of aggregation with SM sensitivity, not possible until the recent development of SM mass photometry, present a timely and exceptional opportunity to make significant advances in a decades-old problem with relevance to biology, physics, chemistry, and medicine. Without this instrumentation, we will not be able to pursue such exciting science.

蛋白质折叠成正确功能所需的复杂结构。这些结构涉及无法总是满足的沮丧的互动，从而允许形成功能障碍的替代结构。实际上，许多疾病与这种错误折叠蛋白的骨料有关。了解聚合如何与本机折叠竞争将产生关键见解，以了解折叠方式如何出现问题，并改善设计新蛋白质的原则。我们将使用干涉光散射探测聚集，以测量溶液中单蛋白的质量。由于此方法对单分子敏感，因此可以监测聚集的少聚集的最早步骤，其中覆盖了聚集级联的所有阶段的异质混合物；因为它是无标签的，所以可以探测聚集，而不会产生通常用于检测的荧光染料的任何偏差。我们将使用单分子（SM）质量光度法来区分不同尺寸（二聚体，三聚体）的低聚物，并分类其形成/生长速率，首次获得集合级联中每个状态的无偏定量度量，以与物理模型进行比较。我们将探测2个具体目标：*** 1。当条件变化时，蛋白质如何从天然转换为汇总。我们将重点介绍tau蛋白：它通常有助于稳定神经元中的微管，但切换到阿尔茨海默氏病和其他痴呆症中的聚集。我们将比较正常和患病条件下低聚物的形成/生长速率，这适合微观模型，以推断出推动行为变化的机制。*** 2。进化如何塑造天然折叠与聚集之间的竞争。人们认为，进化可以选择平滑的能量景观，从而可以快速形成最小的沮丧结构。为了研究这个问题，我们将（a）比较来自不同进化时期的祖先蛋白的聚集，重点是PRP，PRP是一种蛋白质，该蛋白质会引起疾病，并且我们已将其重建的序列重建回约3.5亿年前； （b）测量在计算上设计的蛋白质的聚集，这些蛋白质尚未进行进化选择，即使从头设计越来越可靠，也通常容易汇总。在每种情况下，我们都将比较通过SM质量光度法测量的低聚物的形成/生长速率与天然折叠的特性，例如稳定性，屏障高度和景观粗糙度，我们使用SM力量光谱法测量了我们对折叠的物理变化进行测量，以识别与差异的差异相关的折叠的折叠变化。在与生物学，物理学，化学和医学相关的数十年问题中，取得了重大进展。没有这种仪器，我们将无法追求这种令人兴奋的科学。