Unlocking the intricacies of structures in intrinsically disordered proteins using advanced NMR methods

NMR Spectroscopy offers captivating insights into the world of Intrinsically Disordered Proteins (IDPs) and can help unravel the secrets hidden within seemingly chatic structuress through a focus on dynamics. Birthe B. Kragelund, Professor at the University of Copenhagen, does pioneering work on IDPs using NMR spectroscopy and is dedicated to investigating and understanding these proteins, specifically in the context of membrane proteins, transcription factors, and histone proteins. NMR is key to understanding these dynamic, complex systems.

Birthe studied for her Master’s in chemistry and biochemistry at the Southern Danish University in Odense. During a class covering NMR spectroscopy, she was captivated by the patterns in the spectra. Meanwhile, Birthe also had contact with a research group working with small proteins in lipid metabolism, and these two subjects led her to the Carlsberg laboratory, where she wrote her master’s supervised by Flemming Poulsen, with whom she also wrote her PhD in protein structure and folding.

During her PhD, Birthe spent several months at Oxford University in Chris Dobsons laboratory, studying the kinetics of protein folding. It was during this time that triple resonance was invented, making it possible to label proteins with 15N- and 13C expanding the pattern into more dimensions. Birthe made a decisive turn and went to Lund for a postdoc, where she was taught all the triple resonance NMR spectroscopy techniques, still relevant today.

After her postdoc, Birthe returned to Denmark, where Flemming Poulsen had moved to the University of Copenhagen. Birthe joined there as an Assistant Professor and has stayed at the Department of Biology, today as a full Professor.

In the early 2000s, Birthe’s focus shifted to working with a “new” group of proteins identified through sequencing the human genome, the so-called intrinsically disordered proteins – IDPs. These proteins lack a defined structure, and their NMR spectra were markedly different, posing a fundamental challenge correlating their (lack of) structure to their function.

These disordered yet functional proteins garnered attention within the scientific community. Birthe’s interest was ignited by their ability to discern hidden patterns amid the apparent chaos, akin to the patterns observed in the NMR spectra.

Subsequently, efforts were directed into understanding the features and properties characterizing IDPs. The synergy between NMR spectroscopy and IDP proved to be a match made in heave. Despite the large and disordered nature of these proteins, their dynamic behavior gives sharp and distince peaks in the spectra. Unlike the broad peaks commonly observed in large proteins, the distinctive characteristics of IDPs allowed for a more precise analysis of the spectra.

REPIN Center

Currently, Birthe and her group are directing their studies towards these IDPs, constituting the core research area of the REPIN center, of which Birthe is the head. The establishment of this center was a collaborative effort connecting 5 reaserch groups, at home and internationally. Joining seleveral biochemical and biophysical methodologies. Their center was is buildt on a large grant from the Novo Nordisk Foundation and was initiated in 2019 and is slated to continue its operations until 2027.

A collection of NMR spectrometers is housed by the structural biology and NMR Laboratory (SBiNLab) in the basement of the Department. Initially, with Flemming Poulsen’s move from the Carlsberg Laboratory to UCPH he secured a grant of 27 million DKK from the John and Birthe Meyer Foundation to estabilish the NMR facility. This financial backing facilitated the acquisition of a 750 MHz and 800 MHz instrument. Recognizing the need for technological upgrades in 2013, the Villum Foundation provided a grant for a new 600 MHz instrument with cryo probes. Moreover, funds were assigned to augment the capabilities of the existing 750 MHz instrument by integrating a cryo probe. These advancements underscore the commitment to maintaining state-of-the-art equipment for cutting-edge research.

In 2019, SBiNLab pursued a new 800 MHz instrument from the Novo Nordisk Foundation opening up for the facility by establishment of cOpenNMR. Concurrently, given the numerous applications of NMR spectroscopy still unfamiliar to many, SBiNLab aimed to raise awareness of NMR spectroscopy across different scientific disciplines. To achieve this, twenty percent of the instrument runtime are allocated for external users. A notable initiative is the Daisy award, enabling individuals with no prerequisite of prior NMR knowledge to apply for free NMR time.

A noteworthy project involved collaboration with the Retsmedicinsk Institute. Their aim was to determine the time of death from the metabolite of the eye fluids. While precision is high within 24 hours of discovering the body, it becomes challenging beyond that timeframe. In this context, NMR spectroscopy emerged as a novel approach, highlighting its versatility also for forensic science.

Group and uniqueness

Birthe’s group is sizeable and diverse, thriving on both the center’s activities and her collaborative approach. Comprising 15-20 individuals, including PhDs, postdocs, and a crystallography expert, the team integrates various methodologies within the realm of integrative structural biology. When IDPs bind to partners, they may fold into a structure, allowing isolation, crystallization, and structural modeling.

Despite her substantial contributions to Danish NMR spectroscopy, Birthe doesn’t see herself as a developer but rather as a user, often combining NMR with other techniques. While the field has matured since its competitive early years, specialized developers refine hardware, and a growing community of NMR users, including Birthe, focuses on applying new techniques to scientific challenges rather than contrinutiing to technology development.

Within DANNMR, specialists witness the convergence of different NMR fields. At DTU, Charlotte’s focus on carbohydrates extends to proteins, creating a natural link. In Aarhus, Thibault Viennet’s NMR-oriented work on IDPs complements Birthe’s pursuits. Thus, synergies exist across the country, and there are much to harvest.

Birthe collaborates extensively with cell biologists, aiming to understand how insight into the ensembles of IDPs extends to biology. Her key collaborators include those in REPIN, in SBiNLab and at BIO, and computatioal chemists Kresten Lindorff-Larsen and Robert Best. Additionally, her collaborative efforts extend to industry partners, such as Novo Nordisk and LEO Pharma, where she supervises a PhD alongside Flemming Hofmann. This collaborative network further enriches the dynamic and interconnected community she has cultivated.

Unique NMR methods for understanding IDPs

One of the most captivating projects Birthe has undertaken is that of histone H1 and its chaperone prothymosine alpha (PTMA) . As DNA folds, it forms nucleosomes, and between these nucleosomes exists a linker histone, known for locking them together. These histones, with a small globular domain and a long disordered tail, are highly positively charged binds strongly to DNA. PTMA removes and redistribute H1, making the DNA readable. Notably, PTMA is an IDP and carries a high negative charge.

In collaboration with Ben Schuler and Robert Best, they employed NMR, single-molecule fluorescence, and computational models to study this complex. Astonishingly, both proteins were discovered to be disordered and dynamic in the complex, marking a novel revelation. The binding strength between these two highly dynamic proteins was remarkably high. Subsequent research explored their separation mechanism, revealing that increased concentrations lead to trimer formation, resulting in excelerated exchange rate, effectively separating them. Since 2016, Birthe and her collaborators have continuously uncovering new insights. Currently, they are exploring innovative NMR methods to gain a deeper understanding of phase-separated condensates, experimenting with new NMR types optimized for these samples.

When examining disordered proteins, their binding kinetics often hinder the observation of the bound state due to signal spreading. CEST NMR experiments offer a solution by tracking signals along a frequency. If a visible signal transitions to another state during measurement and subsequently returns, another signal from the bound condition emerges. This provides access to the population, chemical shift, and hence structure of that invisible state. Also the CPMG experiment can be used to access invisible states and is used broadly in NMR spectroscopy.

DANNMR organization

The DANNMR organization originated from the former national center at Carlsberg, initiated by the former generation of NMR scientist. However, as individuals retired and the Carlsberg center closed, it became evident that a reorganization was necessary. Unlike crystallography or Cryo EM, NMR spectroscopy lack a large user group, prompting the need for enhanced communication and visibility in the scientific community.

In response, all deans from technical and natural science universities united, signing a document to establish and mandate this group. Together with Jørgen Skibsted at Århus, Birthe heads DANNMR. The primary goals of the DANNMR consortium include increasing the visibility of NMR spectroscopy in Denmark, ensuring the continuous development of equipment, and educating the next generation to proficiently use and understand NMR tools and methods. Acknowledging the significance of NMR spectroscopy not only in universities but also in industry, the consortium stresses the dissemination of its utility and the provision of training to industry professionals.

Efforts toward these goals involve annual meetings, courses, and a dedicated website to enhance visibility and collaboration (www.dannmr.dk). Although DANNMR plays a crucial role in disseminating NMR awareness, the consortium acknowledges the need for stronger industry ties and strives to encourage companies to adopt NMR spectroscopy. Additionally, discussions within DANNMR have explored the prospect of acquiring a much more powerful magnet, offering superior signal distribution and sensitivity. Collaborative initiatives are under consideration.

The overarching priority of DANNMR is to formulate a comprehensive roadmap, strategically planning and determining optimal resource allocations. This strategic planning will ensure the effective utilization of funds to propel NMR spectroscopy forward in Denmark and beyond.

Strength of Danish NMR

“One of the greatest strengths of the Danish NMR society lies in the long tradition of mutual support and collaboration on larger projects and initiative, such as large national centers. These centers were only possible because of a joint effort towards a common goal, and we need to continue doing this.“

“Perhaps the greatest strength in Danish NMR spectroscopy lies in this historical support and collaboration across various disciplines of science. This diversity within the Danish NMR society is crucial, ensuring that you can always find someone with a different viewpoints on NMR spectroscopy to solve your problem.“