New development makes tiny structural changes in biomolecules visible
FRANKFURT. Even more detailed insights into the cell will be possible in future with the help of a new development in which Goethe University was involved: Together with scientists from Israel, the research group led by Professor Harald Schwalbe has succeeded in accelerating a hundred thousand-fold the nuclear magnetic resonance (NMR) method for investigating RNA.
In the same way that a single piece of a puzzle fits into the whole, the molecule hypoxanthine binds to a ribonucleic acid (RNA) chain, which then changes its three-dimensional shape within a second and in so doing triggers new processes in the cell. Thanks to an improved method, researchers are now able to follow almost inconceivably tiny structural changes in cells as they progress – both in terms of time as well as space. The research group led by Professor Harald Schwalbe from the Center for Biomolecular Magnetic Resonance (BMRZ) at Goethe University has succeeded, together with researchers from Israel, in accelerating a hundred thousand-fold the nuclear magnetic resonance (NMR) method for investigating RNA.
“This allows us for the first time to follow the dynamics of structural changes in RNA at the same speed as they occur in the cell,” says Schwalbe, describing this scientific breakthrough, and stresses: “The team headed by Lucio Frydman from the Weizmann Institute in Israel made an important contribution here.”
The new types of NMR experiments use water molecules whose atoms can be followed in a magnetic field. Schwalbe and his team produce hyperpolarized water. To do so, they add a compound to the water which has permanently unpaired electron radicals. The electrons can be aligned in the magnetic field through excitation with a microwave at -271°C. This unnatural alignment produces a polarization which is transferred at +36°C to the polarization of the hydrogen atoms used in the NMR. Water molecules polarized in this way are heated in a few milliseconds and transfered, together with hypoxanthine, to the RNA chain. The new approach can in general be applied to observe fast chemical reactions and refolding changes in biomolecules at atomic level.
In particular the imino groups in RNA can be closely analyzed using this method. In this way, the researchers were able to measure structural changes in RNA very accurately. They followed a small piece of RNA from Bacillus subtilis, which changes its structure during hypoxanthine binding. This structural change is part of the regulation of the transcription process, in which RNA is being made from DNA. Such small changes at molecular level steer a large number of processes not only in bacteria but also in multicellular organisms and even humans.
This improved method will in future make it possible to follow RNA refolding in real time – even if it needs less than a second. This is possible under physiological conditions, that is, in a liquid environment and with a natural molecule concentration at temperatures around 36 °C. “The next step will now be not only to study single RNAs but hundreds of them, in order to identify the biologically important differences in their refolding rates,” says Boris Fürtig from Schwalbe’s research group.
Publication: Mihajlo Novakovic, Gregory L. Olsen, György Pintér, Daniel Hymon, Boris Fürtig, Harald Schwalbe, Lucio Frydman: A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR, PNAS, 16 January 2020 https://doi.org/10.1073/pnas.1916956117
A picture can be downloaded from: http://www.uni-frankfurt.de/84996281
Caption: Frankfurt researchers followed the movements of this tiny molecule – just two-thousandths of the thickness of a piece of paper. The RNA aptamer changes its structure when it binds hypoxanthine. The green nucleobases change shape particularly quickly, the ones coloured blue more slowly. The grey regions do not change.
Further information: Professor Harald Schwalbe, Center for Biomolecular Magnetic Resonance (BMRZ), http://www.bmrz.de/, Institute of Organic Chemistry and Chemical Biology, Riedberg Campus, Tel.: +49(0)69-798-29737 or -40258, e-mail: firstname.lastname@example.org.
Reported reduction of HFC-23 did not happen
FRANKFURT. According to the two main producers – China and India – the release of the potent greenhouse gas HFC-23 into the atmosphere should have almost completely stopped by 2017. However, the reality is that a team of atmospheric researchers led by the University of Bristol has measured record levels. Dr Kieran Stanley, lead author of the study published in the current issue of “Nature Communications", has been working at Goethe University for six months.
Over the past two decades, researchers have monitored the concentration of the hydrofluorocarbon HFC-23 very closely. “It is a very potent greenhouse gas: The emission of one tonne of this substance does just as much damage as the emission of 12,000 tonnes of carbon dioxide," says atmospheric researcher Professor Andreas Engel from Goethe University. HFC-23 primarily occurs as an unwanted by-product in the manufacture of the refrigerant HCFC-22.
In 2015 India and China, which are considered the main emitters, announced ambitious plans to abate their factory emissions and in 2017 they reported that almost no more HFC-23 was being vented to the atmosphere. This would mean that emissions of this greenhouse gas into the atmosphere between 2015 and 2017 ought to have shown a 90 percent reduction. However, as the international team now reports, emissions have risen further and in 2018 reached an all-time high.
The reduction of HFCs is part of the Kigali Amendment to the Montreal Protocol agreed in 2016. It entered into force in January 2020. Although China and India have not ratified the Amendment, by their own account they had achieved a massive reduction in emissions. “Our study indicates that China has not managed to reduce HFC-23 as reported," concludes Dr Kieran Stanley, who conducted the measurements at the University of Bristol in the framework of the international AGAGE measurement network. Additional measurements will show whether India has successfully implemented its abatement programme.
“This is not the first time there's been controversy about HFC-23 emissions," says Kieran Stanley ruefully. With the United Nations Framework Convention on Climate Change, between 2005 and 2010 the industrial nations created incentives for emerging countries to reduce their emissions. Although emissions of this hazardous greenhouse gas did indeed decrease during that period, the system backfired: Manufacturers did not optimize their processes but instead produced more harmful by-products in order to pocket more funds for destroying them.
The Institute for Atmospheric and Environmental Sciences at Goethe University, where Kieran Stanley is now working as a postdoctoral researcher, has measured a large number of halogenated trace gases at its Kleiner Feldberg measuring station at regular intervals since 2013. Since recently, these measurements are part of the AGAGE network.
Publication: K. Stanley, D. Say, J. Mühle, C. Harth, P. Krummel, D. Young, S. O'Doherty, P. Salameh, P. Simmonds, R. Weiss, R. Prinn, P. Fraser and M. Rigby: Increase in global emissions of HFC-23 despite near-total expected reductions, in Nature Communications, https://doi.org/10.1038/s41467-019-13899-4
Further information: Dr Kieran Stanley, Institute for Atmospheric and Environmental Sciences, Riedberg Campus, Tel.: +49(0)69-798-40249; email@example.com
Single-molecule microscopy visualises the dance of receptors
FRANKFURT. Whether a sick cell dies, divides, or travels through the body is regulated by a sophisticated interplay of signal molecules and receptors on the cell membrane. One of the most important molecular cues in the immune system is Tumour Necrosis Factor α (TNFα). Now, for the first time, researchers from Goethe University have visualised the molecular organisation of individual TNFα receptor molecules and the binding of TNFα to the cell membrane in cells using optical microscopy.
Before TNFα can bind to a membrane receptor, the TNFR receptor must first be activated. By doing so, the key will only fit the lock under certain circumstances and prevents, among other things, that a healthy cell dies from programmed cell death. “For TNFR1 in the membrane, the binding of TNFα is mediated through several cysteine-rich domains, or CRDs," explains Sjoerd van Wijk form the Institute for Experimental Cancer Research in Paediatrics and the Frankfurt Stiftung für Krebskranke Kinder at Goethe University.
In particular, CRD1 of the TNFR1 makes it possible for TNFα to “attach". Researchers already knew that TNFR1 molecules cluster in a fashion similar to a dance, in which two, three or more partners grasp hands – with the dimers, trimers or oligomers consisting of single TNFR1 molecules – in the case of TNFR1. This kind of “structural reorganization" also takes place when there is no TNFα present. “Despite the significance of TNFα for many diseases, including inflammation and cancer, the physiology and patterns of TNFR1 in the cell membrane still remain largely unknown up to now," says Sjoerd Van Wijk, explaining the starting point for his research.
In order to understand the processes in the cell membrane in detail, van Wijk approached Mike Heilemann from the Institute for Physical and Theoretical Chemistry at Goethe University. Using a combination of quantitative microscopy and single-molecule super-resolution microscopy that he developed, Heilemann can visualise individual protein complexes as well as their molecular organisation in cells. Together with Ivan Dikic (Institute for Biochemistry II) and Simone Fulda (Institute for Experimental Cancer Research in Paediatrics) from Goethe University, Harald Wajant from the University Hospital Würzburg and Darius Widera from University Reading/UK, they were able to observe the dance of the TNFα receptors. Financial support was provided by the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Centre 807 “Transport and Communication across Biological Membranes".
As the researchers report in the current issue of “Science Signalling", membrane TNFR1 receptors exist as monomers and dimers in the absence of TNFα. However, as soon as TNFα binds TNFR1, receptor trimers and oligomers are formed in the membrane. The researchers also found indications for mechanisms that determine cell fate independently of TNFα. These findings could be relevant for cancer or and inflammatory diseases such as rheumatoid arthritis. “It clearly opens new paths for developing novel therapeutic approaches," states van Wijk.
Publication: C. Karathanasis, J. Medler, F. Fricke, S. Smith, S. Malkusch, D. Widera, S. Fulda, H. Wajant, S. J. L. van Wijk, I. Dikic, M. Heilemann, Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells. Sci. Signal. 13, eaax5647 (2020). DOI: 10.1126/scisignal.aax5647
Further information: Dr Sjoerd van Wijk, Institute for Experimental Cancer Research in Paediatrics, Niederrad Campus, Tel.: +49 69 67866574, Email: firstname.lastname@example.org
Prof Mike Heilemann, Institute for Physical and Theoretical Chemistry, Riedberg Campus, Tel.: +49 69 798 29424, Email: email@example.com
In ruminants, a bacterium reacts to fluctuating sodium content with two different respiratory circuits
FRANKFURT. Cows can adapt themselves to a fluctuating sodium content in their feed. How they do that was so far a secret. Researchers from Goethe University have now discovered a bacterium in the microbiome of the rumen which has a new type of cell respiration.
The cow can only process grass in its rumen with the help of billions of microorganisms. An entire zoo of bacteria, archaea and protozoa works there like on a production line: First of all, these single-cell organisms break down the cellulose, a polysaccharide. Other bacteria ferment the sugars released into fatty acids, alcohols and gases, such as hydrogen and carbon dioxide. Finally, methanogenic archaea transform these two gases into methane.
An average cow produces about 110 liters of methane per day. It escapes from its mouth through rumination, but also mixes again with partly digested food. As a result, the sodium content of the grass pulp can fluctuate to a considerable degree (between 60 and 800 millimoles of sodium chloride (NaCLl) per liter).
A German-American research team has now discovered how the ruminal bacteria adapt to these extreme fluctuations in sodium content: “Bioinformatic analyses of the genome of ruminal bacteria led our American colleague Tim Hackmann to assume that some ruminal bacteria have two different respiratory circuits. One of them functions with sodium ions and the other without," explains Professor Volker Müller from the Department of Molecular Microbiology and Bioenergetics at Goethe University. That is why Müller suggested to his doctoral researcher Marie Schölmerich that she study a typical representative in the microbiome of ruminants: the bacterium Pseudobutyrivibrio ruminis.
Together with undergraduate student Judith Dönig and Master's student Alexander Katsyv, Marie Schölmerich cultivated the bacterium. Indeed, they were able to corroborate both respiratory circuits. As the researchers report in the current issue of the Proceedings of the National Academy of Sciences (PNAS), the electron carrier ferredoxin (Fd) is reduced during sugar oxidation. Reduced ferredoxin drives both respiratory circuits.
respiratory circuit comprises the enzyme complex Fd:NAD oxidoreductase (Rnf complex). It
uses energy to transport sodium ions out of the cell. When they re-enter the
cell, the sodium ions trigger an ATP synthase, so that ATP is produced. This
respiratory circuit only works in the presence of sodium ions.
In the absence of sodium ions, the bacterium forms an alternative respiratory circuit with another enzyme complex: The Ech hydrogenase (synonymous: Fd:H+ oxidoreductase) produces hydrogen and pumps protons out of the cell. If these re-enter the cell via a second ATP synthase that accepts protons but not sodium ions, ATP is also produced.
“This is the first bacterium so far in which these two simple, completely different respiratory circuits have been corroborated, but our bioinformatic analyses suggest that they are also found in other bacteria," explains Marie Schölmerich. “It seems, therefore, that this adaptation strategy is more widespread," she assumes.
Interestingly, both enzyme complexes (Rnf and Ech) were also discovered in bacteria which are old in terms of evolutionary biology. Professor Müller's research group has examined them in depth, but always only found one of the two enzyme complexes and never both together. “We're now going to use synthetic microbiology methods to produce hybrids of bacteria that contain both complexes in order to optimize them for biotechnological processes. In this way, we can raise the cellular ATP content, which will make it possible to produce products of a higher quality," explains Professor Müller. The intention is to use the respiratory circuits to recover valuable substances through the fermentation of synthesis gas. This is the subject of the trials being conducted in the framework of a project sponsored by the Federal Ministry of Education and Research.
A picture can be downloaded under: https://www.muk.uni-frankfurt.de/84412971?
Caption: The bacterium Pseudobutyrivibrio ruminis (green), a typical ruminal bacterium, obtains energy via two different respiratory circuits. The one requires sodium ions, the other hydrogen ions (H+). In this way, it can adapt to fluctuating sodium concentrations in animal feed in an optimum way.
Picture: Goethe University/ Cow: Shutterstock
Publication: Schölmerich, M.C., Katsyv, A., Dönig, J., Hackmann, T., Müller, V. (20XX). Energy conservation involving two respiratory circuits. Proc. Natl. Acad. Sci. U.S.A., in press.
Further information: Professor Volker Müller, Molecular Microbiology and Bioenergetics, Riedberg Campus, Tel.: +49(0)69-798-29507; VMueller@bio.uni-frankfurt.de.
With the Centre for Biomolecular Magnetic Resonance, Goethe University is one of 23 European partners in the project iNEXT-Discovery
FRANKFURT. Determining the structure of large biomolecules is critical to many innovations in the fields of health, environment and sustainable technologies. Because structural research requires expensive equipment such as NMR spectrometers, the European Union funds research infrastructure. Beginning in February 2020, an additional € 10 million will be invested in the project iNEXT Discovery. The Centre for Biomolecular Magnetic Resonance (BMRZ) at Goethe University is a part of the project once again.
Currently, the iNEXT Collaboration is made up of 23 partners from 14 European countries. It is the first research infrastructure project combining different structural biological methods: X-ray spectroscopy, nuclear magnetic resonance spectroscopy (NMR), electron microscopy and biophysical methods. These methods make it possible to decode the three-dimensional structure of biological macromolecules in order to understand their function within the complex machinery of life. The goal is to develop new medicines, improved vaccinations, new biomaterials, biofuels, and enzymes for food production.
BMRZ at Goethe University makes its expertise in NMR spectroscopy available to researchers throughout Europe. Visitors from other countries already use the equipment daily to determine the structures of proteins, RNA and DNA. It is furthermore possible for industrial partners to participate via cooperation contracts in order, for example, to search specifically for active substances. Training programmes will be set up in the next four years for researchers with little previous experience with NMR.
“At BMRZ, we give European scientists access to the currently most powerful NMR technologies. In the next funding period, a 1.2 gigahertz NMR spectrometer will be available," says Professor Harald Schwalbe, Board Member of iNEXT-Discovery. “From 2020 onwards, we expect that 20 user groups annually will come from all over Europe to use our equipment and profit from our experience. In this way, we are all contributing to exciting science."
Further information: Professor Harald Schwalbe, BMRZ, Institute for Organic Chemistry and Chemical Biology, Tel.: +49-69-798-29737; Email: firstname.lastname@example.org