Proteins are the building blocks of life itself. Understanding the mystery of these complex molecules is something that has perplexed scientists for centuries. Professor Kaare Teilum is one of these scientists dedicated to investigating the fundamental questions of biology from a structural point of view. In his group, they use nuclear magnetic resonance (NMR) to better understand the function and structure of proteins, which could help understand some of the most devastating diseases of present time.
Kaare Teilum’s research focuses on structural biology assisted by NMR spectroscopy. They mostly use NMR spectroscopy to map protein dynamics and for looking at entropy changes, but they
also use it to understand larger structural changes as when ligands bind. Kaare is leading a group of around eight members at any given time including PhD, masters and bachelors’ students and a laboratory technician, and shares on top of that a couple of PhDs and Post-Docs with other groups in Denmark and Sweden.
“Being a part of a group in an environment where you can discuss research is an important part of being a scientist, but it does take up a lot of one’s time managing many students.” Kaare says. Because of this, Kaare has assigned a couple of weeks each summer working alone on an experiment he finds exciting, a way to maintain his passion for science and put aside some of the more stressful day to day tasks.
Kaare started his journey studying biochemistry at the University of Copenhagen (UCPH). When he finished his masters’ degree, he got employed at the Carlsberg Laboratory as a research assistant for the late Professor Flemming Poulsen. It was here he started using NMR to study protein folding, a subject where he and Flemming shared a deep interest. After working a year at Carlsberg, Flemming Poulsen got offered a position as professor at UCPH. Receiving an offer of a PhD stipendium there himself, Kaare decided to follow along.
Not all time during his PhD was spent at UCPH. Kaare also spent several months in Philadelphia with Professor Heinrich Roder, applying continuous flow mixing, a similar but not as developed technique as stopped flow mixing. One of the things he remembered fondly about his trip abroad was the use of non-standardized equipment, which meant he got to spent time working and developing new equipment in a workshop himself.
Although engineering is not Kaare’s main field, he was inspired by his time in Philadelphia and is always trying to push the limits of current experiments. “To understand NMR relaxation, you must understand that in NMR spectroscopy you measure the nuclear spin. And to measure it you must first get the nucleus out of equilibrium; this is usually done by radiating the sample with radio waves and perturbing the spin.” Kaare explains.
The nucleus of an atom can be compared to a small bar magnet, which can be rotated. When a sample is in equilibrium the spins point in the same direction as the magnetic field, but when the spins are perturbed, they go out of equilibrium and will try to move back towards it, a process highly dependent on molecular dynamics. “To stimulate this spin, so that it can return to equilibrium, you will need the NMR spectrometer to have the same frequency as the molecular vibrations, so if you use a 600 MHz spectrometer, the molecules need to have vibrations at 600 MHz as well to return equilibrium.” Kaare elaborates. This method is called NMR relaxation and from these relaxation measurements you can get information about the vibrations in the molecule. “Since the vibrations in the proteins we look at fits perfectly with the NMR frequencies, we can get atomic resolution, which cannot be obtained by most other techniques and this can give information about each proton and carbon nuclei, a very valuable information for understanding the structure.” Kaare tells about the importance of this method. If you want to look at larger proteins you can insert specific probes that only report on specific sites in the protein, but when looking at smaller proteins, you would usually like to get an overview of the entire molecule.
After Kaare finished his PhD, he went to Lund in Sweden as a Post-Doc to continue his studies of dynamical processes in proteins. Here, NMR was an important tool as well, mainly used to give an extra layer of functional understanding of the proteins, in particular they tried to work out why the proteins were not always functioning as they should. In Lund, Kaare looked at the protein Super Oxide Dismutase (SOD) and how it was related to the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). Some cases of ALS were known to be caused by precipitations of SOD and here NMR relaxation was used to observe the dynamic movements that exposed and destabilized the protein giving rise to precipitations, which in turn would cause ALS.
So, NMR can be used to look at how proteins are destabilized, but oppositely it can also be used to observe how proteins are stabilized. “It is easy to destabilize a protein by removing a small part from it, but it is much harder to stabilize it again as a newly inserted stabilizing group would have to fit into the structure perfectly and it can be very hard to predict exactly how to do this since the protein packing needs to be optimal.” Kaare explains. When a structure becomes more compact and stable its vibration declines as it loses entropy, and it wins enthalpy. Oppositely, when you let go of enthalpic interactions the structure by mutations, it gains more dynamics and a higher entropy. “It’s about finding the sweet spot with perfect stability without losing to many dynamics.” Kaare remarks.
Another one of Kaare’s studies investigate the dynamics and stability of mutants of the protein CI2. Here they have found that a general increase of the dynamics can lead to an increase in stability as well – a kind of entropic reinforcement. Instead of a localized change in stability, they see a diffuse change, meaning the whole structure stabilizes. An increase which seems almost unnoticeable when looking at it as it is spread out, but evident when diving deeper into the structure and dynamics.
Billion years old retroviruses
A recent focus of Kaare’s research is within retroviruses, which are a form of virus that inserts a DNA copy of its genome inside a host, thereby changing the genome of that cell to itself. Around 8% of the human genome is leftovers from ancient retroviral infections originating long before we evolved to humans, most of it has mutated into nonsense, but some of it has changed into proteins that
have now gained various functions inside our body. There are around a hundred of these functional proteins originating from old retroviruses left in the body, some billions of years old, others so new that not even all humans contain them. One example is the retroviral Gag proteins, which are well known from HIV. Gag proteins create a shell around HIV RNA, inside the viral particles, which is vital for the HIV virus’ ability to assemble, mature and infect cells.
One of the proteins Kaare’s group is working with is the Activity-Regulated Cytoskeleton-associated (Arc) protein, which is homologous to the retroviral Gag proteins. This Arc protein is used for regulating our nerve cells, which means it is also able to create a protein ball and packs its RNA inside this ball like the Gag protein. If Arc is over-expressed in nerve cells it packs itself with its RNA into extracellular vesicles, that can enter other nerve cells. Scientist do not yet know why Arc does this and what exactly it does functionally, but the fact that it exists means it has some sort of function and that it what Kaare and his are trying to figure out. “These Arc proteins form capsids, a type of ball-like protein shell, but as they are too large for NMR spectroscopy, we cannot use NMR to effectively look at them, instead we use NMR to look at the structures of the Arc monomers individually and then we observe what happens when they react with their neighbors creating oligomers. This is the process by which capsids are made, this way we get information about capsids without looking at them directly.” Kaare explains.
In some of his newer projects Kaare is aiming for more of an application perspective, where his former
projects have been mostly basic research. Currently, he is investigating capsids as a possible drug delivery system for small as well as macro molecules and how to deliver them to cells without activating the immune system. In another project he looks at phosphate binding proteins, which can be used to remove phosphate from wastewater, rivers, and lakes with high phosphate pollution and reuse the material. Current methods to remove phosphate, obtain impure products, which are unfavorable since they are often reused as fertilizer, which requires it to be in a pure form. Using proteins instead for capture and release can produce very pure phosphate from creeks and streams compared to other inorganic sorbents and they can even be used to extract the proteins from inorganic sorbents as well.
“The reason we started looking at these proteins was that we had developed a system for optimization of protein stability. We then considered where stable proteins could be beneficial. In wastewater billions of liters of water flow across these phosphate removal proteins putting them under immense pressure. We then realized that if they could be stabilized, they could be so much more effective.” Kaare explains. With this, Kaare also wanted to show that their understanding of protein stabilization could be used in something assisting in creating a sustainable future.
Strength of Danish NMR
In his phosphate removal project, he is already working with a chromatography company in Kastrup called Upfront Chromatography led by Dr. Kenneth Harlow, where they have developed a technology that may be used for impure samples of wastewater. Besides, Kaare is also collaborating with researchers at other departments at the University of Copenhagen, to try to understand the amyloid beta peptide which precipitates in fibrils in the brains of people with Alzheimer’s and figuring out how metal ions have a significance within this. Exactly these possibilities of collaboration with other fields, but also within the Danish NMR society are one of the things Kaare sees as a strength of Danish NMR. “The strength of Danish NMR is that we cover wide grounds with a breadth of unique groups and ideas, but despite being so different we are good at integrating our research and helping each other forward.” Kaare says about the strength of Danish NMR.
Written by: Jonatan Emil Svendsen