Periodic Reporting for period 4 - F-BioIce (Fundamentals of Biological Ice Nucleation)
Periodo di rendicontazione: 2023-10-01 al 2025-03-31
Ice nucleating bacteria attack plants by growing ice crystals with specialized ice-nucleating proteins (INPs) that are present at their outer cell walls. INPs promote ice crystallization at high subzero temperatures and cause severe frost damage in plants. Large numbers of ice bacteria are emitted into the atmosphere, where they influence ice formation, clouds and rainfall. Under atmospheric conditions, pure water will not freeze above -40° C. Soot and dust typically elevate the nucleation temperature to around -20° C. Airborne INPs can bring the nucleation temperature near -5° C. INPs are the most efficient ice nucleators known. Such efficient ice growth has tremendous impact on our climate. The Report of the Intergovernmental Panel on Climate Change concludes that the role and mode of action of biological IN is currently unclear. It also states, that the role of aerosols is the largest factor of uncertainty for current climate models. It is completely unknown, how ice proteins come together with airborne water droplets at the molecular level. Water and ice are the foundation of life. Yet, we do not know how INPs control water and freezing in ecosystems around the globe. With F-BioIce our goal is to unravel Nature’s tricks that make INPs so much better at nucleating ice than any other material known.
Current applications in snowmaking and food science use trial and error approaches with fragments of ice bacteria. F-BioIce has been providing new structure-based approaches to pave the way towards new technologies that imitate the concepts of bacterial freezing. We hope that such understanding will boost bioinspired innovations in cryopreservation of organs, cryo-therapy, food, cloud seeding and artificial icing and anti-icing surfaces.
Experimental evidence of how INPs manipulate water has been difficult to obtain. Very recent developments in laser-based ultrafast sum frequency generation (SFG) spectroscopy allowed us to probe molecular interfaces with high precision. We have had a strong focus on structural biology at interfaces within F-BioIce and we are now applying new technologies to understand INP action. We made first steps solving the puzzle of biological ice nucleation by determining how INPs are folded at water interfaces. The folding of a protein is key to its function and understanding the structure of ice proteins will allow insights into how water is frozen so effectively. We have also been building techniques to follow how heat flows through the surface of the bacteria to understand whether INPs use a specific trick to funnel heat away from the interface during freezing. Within follow up project of F-BioIce, these methods will be used in the future for a wholistic picture of bacterial ice nucleation and environmental impacts.
Current applications in snowmaking and food science use trial and error approaches with fragments of ice bacteria. F-BioIce has been providing new structure-based approaches to pave the way towards new technologies that imitate the concepts of bacterial freezing. We hope that such understanding will boost bioinspired innovations in cryopreservation of organs, cryo-therapy, food, cloud seeding and artificial icing and anti-icing surfaces.
Experimental evidence of how INPs manipulate water has been difficult to obtain. Very recent developments in laser-based ultrafast sum frequency generation (SFG) spectroscopy allowed us to probe molecular interfaces with high precision. We have had a strong focus on structural biology at interfaces within F-BioIce and we are now applying new technologies to understand INP action. We made first steps solving the puzzle of biological ice nucleation by determining how INPs are folded at water interfaces. The folding of a protein is key to its function and understanding the structure of ice proteins will allow insights into how water is frozen so effectively. We have also been building techniques to follow how heat flows through the surface of the bacteria to understand whether INPs use a specific trick to funnel heat away from the interface during freezing. Within follow up project of F-BioIce, these methods will be used in the future for a wholistic picture of bacterial ice nucleation and environmental impacts.
To determine how INPs are freezing water, we have been asking the question: What interface do ice bacteria present to water surface?
There is no experimental evidence showing how ice proteins come together with water molecules at the molecular level. Within F-BioIce we will attempt to answer the question: What side chains affect the impact on water ordering? What is the structure of water molecules at the surface of INPs? We have been working on the long overdue connection of biological surface spectroscopy and climate research to determine what makes the surface of an ice active protein the best atmospheric ice nucleator known. We have prepared synthetic ice nucleating proteins, in which we have been able to control the amino acid sequence and size of the ice nucleating sites with precision. We then exposed the proteins to a water surface and probed the molecular structure of proteins and water molecules. Monitoring essentially single layers of proteins in contact with a single layer of water molecules is not a trivial task. We succeeded by using a surface spectroscopy, which is based on frequency mixing of infrared and visible laser pulses with the duration of only 40 femtoseconds.
We found that at room temperature the ice proteins were not interacting with the interfacial water to a great extent. However, when the temperature come close to the 'working temperature' of an ice protein, the proteins reorient and fully expose their ice active domains to the surrounding water. This increase water order and, in return, promotes freezing. The ice activity of the proteins is essentially switched on at temperatures that are relevant for biological ice nucleation. A second activation trigger we observed is related to pH. If the water environment becomes slightly acidic, the ice proteins loose their order with respect to each other. To effectively nucleate ice a cell builds large clusters of ice proteins at its surface. If the order is reduced, the clusters are becoming unstable and ice nucleation is less effective. The loss of large ice nucleating clusters has been proposed a mechanism by which the bacterium can activate ice nucleation. These results have been published in scientific articles and international conferences.
There has been the question of how the protein orders water at the interface with ideas going from hydrogen bonding to biomimicry of the basal plane of ice. We have therefore investigated the water interactions of one of the most prominent side chains of INPs, the amino acid side chain threonine. Threonines are special because they carry a hydrophobic and also a hydrophilic group. Using SFG spectroscopy in combination with a new method to calculate water spectra, we determined the interaction of water with threonine in atomic detail. These results are currently under preparation for publication in a scientific journal. Presentation of the results by lead author and then-PhD student Dr. Kris Strunge was awarded the poster prize at the prestigious Gordon Conference for Vibrational Spectroscopy.
There is no experimental evidence showing how ice proteins come together with water molecules at the molecular level. Within F-BioIce we will attempt to answer the question: What side chains affect the impact on water ordering? What is the structure of water molecules at the surface of INPs? We have been working on the long overdue connection of biological surface spectroscopy and climate research to determine what makes the surface of an ice active protein the best atmospheric ice nucleator known. We have prepared synthetic ice nucleating proteins, in which we have been able to control the amino acid sequence and size of the ice nucleating sites with precision. We then exposed the proteins to a water surface and probed the molecular structure of proteins and water molecules. Monitoring essentially single layers of proteins in contact with a single layer of water molecules is not a trivial task. We succeeded by using a surface spectroscopy, which is based on frequency mixing of infrared and visible laser pulses with the duration of only 40 femtoseconds.
We found that at room temperature the ice proteins were not interacting with the interfacial water to a great extent. However, when the temperature come close to the 'working temperature' of an ice protein, the proteins reorient and fully expose their ice active domains to the surrounding water. This increase water order and, in return, promotes freezing. The ice activity of the proteins is essentially switched on at temperatures that are relevant for biological ice nucleation. A second activation trigger we observed is related to pH. If the water environment becomes slightly acidic, the ice proteins loose their order with respect to each other. To effectively nucleate ice a cell builds large clusters of ice proteins at its surface. If the order is reduced, the clusters are becoming unstable and ice nucleation is less effective. The loss of large ice nucleating clusters has been proposed a mechanism by which the bacterium can activate ice nucleation. These results have been published in scientific articles and international conferences.
There has been the question of how the protein orders water at the interface with ideas going from hydrogen bonding to biomimicry of the basal plane of ice. We have therefore investigated the water interactions of one of the most prominent side chains of INPs, the amino acid side chain threonine. Threonines are special because they carry a hydrophobic and also a hydrophilic group. Using SFG spectroscopy in combination with a new method to calculate water spectra, we determined the interaction of water with threonine in atomic detail. These results are currently under preparation for publication in a scientific journal. Presentation of the results by lead author and then-PhD student Dr. Kris Strunge was awarded the poster prize at the prestigious Gordon Conference for Vibrational Spectroscopy.
Protein structure is the basis for protein function. It is therefore important to understand how proteins are folded and structured in order to find out how these machines of life work. For this reason, protein scientist have been determining protein structure with atomic resolution for decades now. With F-BioIce we have developed new tools to probe protein structure at interfaces by combining laser spectroscopy with computer simulations. The result is a structure with high accuracy, which is based on both theory and experiment. Within F-BioIce this methodology was used to determine how ice proteins are folding and orienting at water surfaces. This was a major leap forward since only little experimental data about ice proteins at interfaces had been available before. These methods have also already been applied in related areas to determine how proteins operate. Enzymes breaking down biological surfaces, mineral proteins building cell walls, bone and nanostructure, membrane proteins, amyloidic proteins involved in Alzheimers, the methods we have developed here have been used in these field for a high resolution view of how proteins interact with surfaces. All in all, F-BioIce has brought the community closer to understanding the molecular details of ice nucleation. It has also promoted methods critical for other important areas of research where proteins are interacting with interfaces.