Redefining the niche for protein solution NMR spectroscopy: Side chain and domain motions

目的我们正在开发核磁共振波谱的新方法，以在原子水平上详细研究蛋白质的结构。高纯度的溶解在水中的蛋白质样品被放置在超导磁体中，并用无线电波进行探测。通过这种方式，我们可以研究给定蛋白质中所有原子的位置和迁移率。背景蛋白是驱动所有生命过程的分子机器。人体内大约有2万种不同的蛋白质，每种蛋白质都由自己的DNA基因编码。最近，谷歌DeepMind使用人工智能方法预测了人类蛋白质组中所有20,000种蛋白质的结构，数据库中包含了100,000‘S通过X射线结晶学解析的蛋白质结构。这项技术将纯化的蛋白质排列成单晶体，然后在用X射线探测之前对其进行超冷却。问题是，蛋白质结构被很好地可视化，但它实际上比“冻结”模型显示的更具流动性。因此，我们正在开发使用核磁共振的技术来告诉我们蛋白质的哪些部分实际上是刚性的，而不是可移动的。方法学1.侧链移动性。氨基酸连接在一起，形成一个蛋白质骨架，侧链伸出。有20个不同的侧链对应着20种不同的氨基酸类型。基因规定了氨基酸的确切顺序和类型。我们正在开发核磁共振方法来确定蛋白质中哪些侧链是刚性的，而不是柔性的。2.域移动性。蛋白质往往折叠成小球，称为区域，每个区域包含50-200个氨基酸。蛋白质结构域之间可以有很大的灵活性，但使用核磁共振很难同时研究多个结构域。因此，我们正在开发核磁共振方法，使同时研究多个结构域成为可能，以便我们能够了解蛋白质结构域如何移动以相互调节。目的我们开发的技术适用于所有蛋白质，但我们将在两个方面进行测试：1.心肌肌钙蛋白复合体在每次心跳时开启和关闭心肌收缩。我们将研究药物起伏时蛋白质侧链是如何运动的。这些药物改变了收缩和放松之间的平衡，以治疗不同类型的心力衰竭。我们还将研究身体如何通过改变肌钙蛋白的不同区域来控制这种平衡。2.像ArlR这样的反应调节蛋白帮助细菌对不同的环境做出反应。这有助于细菌只有在知道自己必须在人类宿主体内生存的情况下才能启动许多有害的基因。我们将研究侧链运动和结构域运动，以更好地了解这些类型的蛋白质是如何开启和关闭的。现在可以在原子水平上看到蛋白质的结构细节。然而，要了解它们是如何真正起作用的，我们必须了解它们是如何运动的。这对任何试图在分子水平上理解生命过程的人来说都非常重要。

Purpose We are developing new methods in nuclear magnetic resonance (NMR) spectroscopy to study the structure of proteins at atomic level detail. Highly purified samples of proteins dissolved in water are placed in a superconducting magnet and probed with radio waves. In this way, we can study the position and mobility of all atoms in a given protein. Background Proteins are the molecular machines that drive all life processes. There are about 20,000 different proteins in the human body, each encoded by its own DNA gene. Recently, Google DeepMind used artificial intelligence methods to predict the structures of all 20,000 proteins in the human proteome using databases that contain 100,000's of protein structures solved by X-ray crystallography. This technique aligns purified proteins into a single crystal and then super-cools it before probing with X-rays. The problem is that the protein structure is well visualized, but it is actually more mobile than the "frozen" model indicates. We are thus developing techniques using NMR to tell us which parts of the protein are actually rigid versus mobile. Methodology 1. Side chain mobility. Amino acids are linked together to form a protein backbone with side chains sticking out. There are 20 different side chains corresponding to the 20 different amino acid types. Genes specify the exact order and type of amino acids. We are developing NMR methods to determine which side chains in a protein are rigid versus flexible. 2. Domain mobility. Proteins tend to fold up into small balls called domains containing 50-200 amino acids each. There can be a lot of flexibility between protein domains, but it is difficult to study more than one domain at a time using NMR. We are thus developing NMR methods that make it possible to study multiple domains at the same time so that we can understand how protein domains move to regulate each other. Objectives The techniques that we develop are applicable to all proteins, but we will test them out on two: 1. The cardiac troponin protein complex turns heart muscle contraction on and off with every heartbeat. We will study how protein side chains move when drugs come on and off. The drugs shift the balance between contraction and relaxation to treat different types of heart failure. We will also study how the body controls this balance by shifting around the different domains of troponin. 2. Response regulator proteins like ArlR help bacteria to react to different environments. This helps bacteria to turn on a lot of nasty genes only when they know they have to survive inside a human host. We will study side chain motions and domain motions to better understand how these types of proteins are switched on and off. Impact It is now possible to see the structures of proteins at atomic level detail. However, to understand how they truly work we have to understand how they move. This is very important to anyone trying to understand life processes at the molecular level.