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

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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.