High-bandwidth laser tweezers detection system for directly observing the transition paths taken by single proteins and nucleic acids during structural transitions

生物分子，如构成我们身体的蛋白质，必须形成复杂的结构才能正常发挥作用。这些结构是如何形成的--这一过程被称为“折叠”--对于理解生物功能和解决由错误结构引起的疾病来说，这是一个重要的问题。然而，折叠的微观细节很难观察到，因为它们非常快，涉及非常微小的运动。我们已经开发出“光学镊子”，它使用激光抓住单个蛋白质分子并将其拉开，使我们能够以原子尺度的分辨率观察结构变化。然而，这些动议的时间尺度仍然太快，无法适当地解决它们。这个项目将使我们能够建造新的镊子仪器，具有足够快的时间分辨率-有史以来第一次-在改变蛋白质结构的同时观察蛋白质。它将为折叠问题打开一扇新的窗口，使我们能够以前所未有的细节研究折叠的微观机制。该仪器将首先应用于结构简单的分子(例如小蛋白质和核酸)，这些分子已经通过其他技术进行了深入的研究，包括实验和计算。通过与以前的结果进行比较，我们将验证测量结果，并展示分辨率的改进。然后，我们将测量参与调控基因表达的具有更复杂结构的分子，以更好地了解结构复杂性对分子在折叠过程中所采取的路径的影响。最后，我们将使用该仪器研究蛋白质，这些蛋白质有时会形成错误的结构，从而导致“疯牛病”和卢·格里克病等疾病。通过表征蛋白质在结构转变过程中所走的路径，我们希望区分正确路径和导致疾病结构的路径的属性。

Biological molecules like the proteins that make up our bodies must form intricate structures to function properly. How these structures form - a process called "folding" - is an important question for understanding biological function and for addressing diseases that arise from incorrect structures. However, the microscopic details of folding are difficult to observe because they are very fast and involve very small motions. We have developed "optical tweezers" that use lasers to grab onto single protein molecules and pull them apart, allowing us to observe structural changes with atomic-scale resolution. The timescale of the motions, however, is still too fast to resolve them properly. This project will allow us to build new tweezers instrumentation with time resolution fast enough - for the first time ever - to observe the protein while in the midst of changing its structure. It will open a new window on the folding problem, allowing us to investigate the microscopic mechanisms of folding in unprecedented detail. The instrument will first be applied to molecules with simple structures (e.g. small proteins and nucleic acids) that have been studied in depth by other techniques, both experimental and computational. By comparing to previous results, we will validate the measurements and demonstrate the improvements in resolution. We will then measure molecules with more complex structures that are involved in regulating gene expression, to understanding better the effects of structural complexity on the pathways taken by the molecule during folding. Finally, we will use the instrument to study proteins that sometimes form incorrect structures, thereby causing diseases such as "mad cow" disease & Lou Gehrig's disease. By characterising the paths taken by the proteins during the structural transitions, we hope to distinguish properties that differentiate the correct paths from those leading to diseased structures.