NMRofLargeComplexes · Unveiling dynamics and substrate interactions of large protein complexes by NMR spectroscopy

Unveiling dynamics and substrate interactions of large protein complexes by NMR spectroscopy

Proteins are biological machines that make life possible. Faulty proteins can lead to diseases and many proteins are targets for drugs. Therefore, it is very important to understand how proteins function. Over the past decades methods have been developed to determine the structure of proteins – an astonishing feat since proteins are typically a billionth of a meter small. However, a structure is frozen in time. Yet to understand protein function, we need to understand how proteins move. Also, we need to know how a protein interact with other molecules. Solution-state nuclear magnetic resonance (NMR) is a scientific method that is particularly well-suited to determine how molecules move and how they interact. However, NMR has one major disadvantage: It can only be applied to small molecules. One might assume that all proteins are small, but that is not actually the case. Some are 3 nanometer in diameter (quite small) others might be bigger than 15 nanometers (quite big). Unfortunately, many proteins that we are interested in are either big or function together with other proteins so that they form what is called a protein complex. Here we show how NMR can be applied to very big protein complexes that consist of many non-identical proteins (asymmetric protein complexes). As our “work horse” protein we chose the RNA exosome. The exosome is a molecular machines that degrades RNA, the building plan of proteins, into small pieces. It consists of 10 distinct proteins. That makes it a giant in the world of proteins. The process of RNA degradation by the exosome is vital for all organisms be it humans, mice or yeast. Faulty exosomes can cause severe diseases in humans. So we are looking at an absolutely essential protein complex. Why then isn’t it possible to look at big protein complexes by NMR? The exosome consists of roughly 55000 atoms and most of them could potentially give rise to an NMR signal – far too many to be manageable. More importantly, the larger a protein, the broader its NMR signals (for quantum mechanical reasons). For large proteins, signals are so broad that they disappear in the noise. We can counter both of these problems with a trick. Proteins consist of 20 different types of building blocks, called amino acids. Recent developments in bioengineering make it possible to produce proteins in such a way that only 1 or 2 types of these amino acids are visible in an NMR experiment. In addition, we can pick 2 amino acids for which the signal broadening problem is not so severe (again quantum mechanics). The procedure is called methyl-labeling. As a result only very few atom groups (typically 10-30) give rise to signals. This is enough to monitor every part of the exosome. Alternatively, we can also incorporate one single fluorine atom into a protein resulting in an NMR spectrum with one single peak. In addition to methyl- and fluorine-labeling techniques, improvements in the software and hardware of NMR spectrometers make it possible to obtain even sharper signals. When all of these recent developments are combined, we can look at individual atoms of molecular giants, like the exosome, and study their motion, interactions with other atoms and their localization inside the exosome. We thus demonstrate how it is possible to study dynamics and molecular interactions of big molecular machines, like the RNA exosome, by NMR. The approach provides information that is complementary to structural studies and thus advances our understanding of how big molecular machines function. This in turn is invaluable to understand how a cell sustains what we call life and to understand what happens when the machines malfunction, i.e. to understand disease.

Data: CORDIS, © European Union