Periodic Reporting for period 1 - NMRofLargeComplexes (Unveiling dynamics and substrate interactions of large protein complexes by NMR spectroscopy)
Reporting period: 2020-09-01 to 2022-08-31
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.
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.
An NMR spectrum contains one or several peaks and it is crucial to know which amino acid gives rise to which peak. This procedure is called assignment and it is a bottleneck step for every NMR-based research project. We individually mutated all Ile and Met amino acids from 3 of the 10 subunits to other amino acids in order to assign resonances.
Next, we compared spectra of Exo9 with spectra of Exo10. We observed that the cap of the exosome does not interact with Rrp44 while subunits in the ring do interact with it, which is manifested by peak shifts in the spectrum. This means that the ring but not the cap binds to the catalytic subunit. Similarly, we observe that all studied subunits show peak shifts upon addition of RNA providing detailed insights into the path that the RNA takes through the exosome.
Next, we identified an extended loop region (ELR) in the exosome, for which we did not have any structural information. We tagged the ELR with a paramagnetic compound. Proximity of the compound to an amino acid leads to reduced peak intensities. We observe no effect for the cap but we see effects for ring subunits. We can conclude that the ELR is far away from the cap but close to the ring. However, in the presence of RNA, peak intensities do not change for any of the subunits suggesting that the loop is displaced from the exosome core by the RNA. In a next step, we studied the dynamics of the ELR. In Exo9 we observe fast dynamics between two conformations of the ELR. In Exo10 the dynamics become slower. When we add RNA the dynamics are almost gone.
To test the functional role of the ELR, we blocked the exosome channel so that the RNA cannot pass through it anymore. The channel-blocked exosome loses all its activity because RNA cannot access the catalytic subunit. When we then removed the ELR from the channel-blocked exosome, we observe activity of the exosome. That implies that RNA can access the catalytic subunit using a pathway that bypasses the channel. In living cells this would potentially be lethal since the channel is needed to select which RNA is to be degraded and which is not. The ELR thus has a valve-like function that allows correctly bound RNA to be degraded and prevents incorrectly bound RNA from being degraded.
The results of this work have been made available to the scientific community as well as a layperson audience. The project was presented at several international scientific conferences. A video was produced that summarizes key findings and a website was designed that is specifically aimed at laypersons. A manuscript is currently in preparation and will be published open-access in a scientific journal in the near future.
Next, we compared spectra of Exo9 with spectra of Exo10. We observed that the cap of the exosome does not interact with Rrp44 while subunits in the ring do interact with it, which is manifested by peak shifts in the spectrum. This means that the ring but not the cap binds to the catalytic subunit. Similarly, we observe that all studied subunits show peak shifts upon addition of RNA providing detailed insights into the path that the RNA takes through the exosome.
Next, we identified an extended loop region (ELR) in the exosome, for which we did not have any structural information. We tagged the ELR with a paramagnetic compound. Proximity of the compound to an amino acid leads to reduced peak intensities. We observe no effect for the cap but we see effects for ring subunits. We can conclude that the ELR is far away from the cap but close to the ring. However, in the presence of RNA, peak intensities do not change for any of the subunits suggesting that the loop is displaced from the exosome core by the RNA. In a next step, we studied the dynamics of the ELR. In Exo9 we observe fast dynamics between two conformations of the ELR. In Exo10 the dynamics become slower. When we add RNA the dynamics are almost gone.
To test the functional role of the ELR, we blocked the exosome channel so that the RNA cannot pass through it anymore. The channel-blocked exosome loses all its activity because RNA cannot access the catalytic subunit. When we then removed the ELR from the channel-blocked exosome, we observe activity of the exosome. That implies that RNA can access the catalytic subunit using a pathway that bypasses the channel. In living cells this would potentially be lethal since the channel is needed to select which RNA is to be degraded and which is not. The ELR thus has a valve-like function that allows correctly bound RNA to be degraded and prevents incorrectly bound RNA from being degraded.
The results of this work have been made available to the scientific community as well as a layperson audience. The project was presented at several international scientific conferences. A video was produced that summarizes key findings and a website was designed that is specifically aimed at laypersons. A manuscript is currently in preparation and will be published open-access in a scientific journal in the near future.
Previously, it was not possible to study large, asymmetric proteins like the RNA exosome and experiments were conducted on relatively small proteins or on symmetric complexes. With our research, we have demonstrated that it is possible to study large, asymmetric protein complexes using solution-state NMR. This allows us to “see” more of the dynamics and interactions happening in the microscopic world of proteins.
Our methodolgy is not only of interest to fundamental biomolecular research but it may also have direct applications relevant for instance for drug discovery where – potentially dynamic – interactions between drugs and proteins are of interest. With the methods developed in this project it is possible to study drug interactions with large proteins by NMR as well as the impact that drug binding has on protein dynamics and structure. Finally, protein dynamics and molecular interactions directly correlate with proper protein function and it is thus of fundamental interest to understand these properties in order to understand life and to cure diseases.
Our methodolgy is not only of interest to fundamental biomolecular research but it may also have direct applications relevant for instance for drug discovery where – potentially dynamic – interactions between drugs and proteins are of interest. With the methods developed in this project it is possible to study drug interactions with large proteins by NMR as well as the impact that drug binding has on protein dynamics and structure. Finally, protein dynamics and molecular interactions directly correlate with proper protein function and it is thus of fundamental interest to understand these properties in order to understand life and to cure diseases.