RUI: Rigorous physical interpretation of vibrational probe frequencies in proteins

了解蛋白质的结构以及这些结构如何随着周围环境的变化而变化，是与生物学、生物化学、生物医学科学和疾病治疗广泛相关的一项挑战。化学家经常使用分光计（测量分子如何吸收、发射或散射光的仪器）来获取分子结构的信息。但由于蛋白质是大分子（数百或数千个原子），而且具有灵活性，因此仅通过光谱学很难确定它们的结构。在这个项目中，由化学学部化学结构动力学和机理（CSDM-A）项目支持，哈弗福德学院的Casey Londergan教授正在使用实验光谱学和计算化学的结合来开发“振动探针”技术，以确定蛋白质的结构和内部运动。振动探针是一种小分子，当它被隔离时，以已知的频率（通常对应于红外光）振动，但当它与其他分子（包括其他探针）相互作用时，频率会发生变化。正在研究的一种这样的探针是硫氰酸盐，它含有硫、碳和氮（SCN）。通过将SCN或其他振动探针连接到蛋白质的不同部分，Londergan教授假设探针频率的变化可能与局部探针环境的变化有关。原则上，振动探针因此报告蛋白质的局部区域及其与蛋白质的其他部分或与其他分子（水，溶解离子等）的相互作用。然而，为了解释实验观察到的探针频率变化，计算机模拟不同的蛋白质形状和局部环境是必要的。这个项目主要由本科生进行，并通过本地和国际合作者的网络为他们提供跨学科的研究经验。该项目还包括教育推广活动，例如以舞蹈为基础的讲习班和指导，旨在提高对蛋白质及其相互作用的人类水平的理解。本研究项目的重点是一种模式调节蛋白（钙调蛋白），一种模式膜蛋白（α -突触核蛋白）和其他蛋白质在细菌生物合成的中心。采用采样良好的分子动力学（MD）模拟来提出结构的集合，然后使用振动探针组进行进一步的模拟，以产生模拟的光谱线形状，从而可以验证或帮助重新加权模拟的结构集合。基于相对广泛使用的SCN探针组，正在开发测量光谱与局部探针环境之间的初始模型关联。随着方法准确性的提高，它最终将用于对该项目将开发的较少使用的探针基团（例如，叠氮基、异腈基、炔基和硝基）的新数据提供定量解释。这些新型探针群中的一些信号更容易用拉曼光谱而不是红外吸收来收集。该项目开发的定量振动探针方法可能能够解决许多重要的生物分子问题，其中当前数据无法确定表示构象分布，这在无序和“模糊”蛋白质系统（其中大多数是调节和/或疾病相关物种）中至关重要。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Understanding the structures of proteins and how these structures change in response to their surroundings is a challenge of broad relevance to biology, biochemistry, biomedical science, and disease treatment. Chemists often use optical spectrometers (instruments that measure how molecules absorb, emit or scatter light)to obtain information about molecular structure. But as proteins are large molecules (hundreds or thousands of atoms) that are also flexible, it is very difficult to determine their structures from optical spectroscopy alone. In this project, supported by the Chemical Structure Dynamics and Mechanism (CSDM-A) program of the Chemistry Division, Professor Casey Londergan of Haverford College is using a combination of experimental spectroscopy and computational chemistry to develop "vibrational probe" techniques for the determination of the structures and internal motions of proteins. A vibrational probe is a small molecule that vibrates at a known frequency (typically corresponding to infrared light) when it is isolated, but exhibits a change in frequency when it interacts with other molecules (including other probes). One such probe being investigated is thiocyanate, which contains sulfur, carbon and nitrogen (SCN). By attaching SCN or other vibrational probes to different parts of the protein, Prof. Londergan hypothesizes that the changes in probe frequencies can be associated with changes in their local probe environment. In principle, the vibrational probes thus report on local regions of the proteins and their interactions with other parts of the protein or with other molecules (water, dissolved ions, etc.). However, in order to interpret the experimentally observed probe frequency changes, computer simulations of the different protein shapes and local environments is necessary. This project is being conducted mainly by undergraduate students and is providing them with an interdisciplinary research experience with a network of local and international collaborators. This project also includes educational outreach activities, for example dance-based workshops and instructions designed to engender a more human-level understanding of proteins and their interactions. This research project is focusing on a model regulatory protein (calmodulin), a model membrane protein (alpha-synuclein), and other proteins at the center of bacterial biosynthesis. Well-sampled molecular dynamics (MD) simulations are employed to propose ensembles of structures, and then further simulations with vibrational probe groups explicitly included to produce simulated spectral lineshapes that can validate or help to re-weight the simulated structural ensembles. The initial model associations between measured spectra and local probe environments are being developed based on the relatively broadly-used SCN probe group. As the methodology improves in accuracy, it will ultimately be used to provide quantitative interpretation of new data from less-used probe groups that this project will develop (e.g.,azido, isonitrile, alkyne, and nitro groups). Some of these novel probe groups' signals are more easily collected by Raman spectroscopy rather than infrared absorption. This project's development of a quantitative vibrational probe methodology is likely to enable solutions to many important biomolecular problems where current data are underdetermined for representing the conformational distribution, which is of central importance in disordered and "fuzzy" protein systems(most of which are regulatory and/or disease-related species).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.