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

蛋白质折叠成正确功能所需的复杂结构。这些结构包括不能总是被满足的令人沮丧的相互作用，允许导致功能障碍的替代结构形成。事实上，许多疾病都与这种错误折叠的蛋白质聚集有关。了解聚合如何与天然折叠竞争，将有助于了解折叠是如何出错的，并改进设计新蛋白质的原则。我们将使用干涉光散射法探测聚集，以测量溶液中单个蛋白质的质量。由于这种方法对单分子敏感，它可以监测聚集的最早步骤——覆盖聚集级联所有阶段的非均质混合物中的小寡聚物；因为它是无标记的，聚集可以探测没有任何偏差的荧光染料通常用于检测。我们将使用单分子（SM）质谱法来区分不同大小的低聚物（二聚体，三聚体），并编目它们的形成/生长速度，首次获得聚集级联中每个状态的无偏定量测量，用于与物理模型进行比较。我们将探讨两个具体目标：***1。当条件改变时，蛋白质是如何从原生转变为聚合的。我们将以tau蛋白为例：它通常有助于稳定神经元中的微管，但在阿尔茨海默病和其他痴呆症中会转变为聚集。我们将比较正常和患病条件下低聚物的形成/生长速度，拟合微观模型来推断驱动行为变化的机制。进化如何塑造原生折叠和聚合之间的竞争。进化被认为是选择平滑的能量景观，允许快速形成最小挫折结构。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质的聚集，重点是PrP，一种聚集导致疾病的蛋白质，我们已经重建了3.5亿年前的序列；(B)测量计算设计的蛋白质的聚集，这些蛋白质没有经历进化选择，即使从头设计越来越可靠，也经常倾向于聚集。在每种情况下，我们将比较SM质谱法测量的低聚物的形成/生长速度与SM力谱法测量的天然折叠特性，如稳定性、屏障高度和景观粗糙度，以确定折叠中与聚集倾向差异相关的物理变化。***这种SM灵敏度聚集的研究，直到最近SM质谱仪的发展才成为可能，为在与生物、物理、化学和医学相关的数十年问题上取得重大进展提供了及时和特殊的机会。如果没有这些仪器，我们将无法从事如此激动人心的科学研究。

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.