Periodic Reporting for period 2 - F-BioIce (Fundamentals of Biological Ice Nucleation)
Reporting period: 2020-10-01 to 2022-03-31
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.34 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. We are going far beyond the current state of the art with new technologies to probe the structure and molecular motions involved in INP freezing action in real time. This information will provide a foundation for researchers to define a new nucleation theory that includes water structure and thermodynamics within two-dimensions, diffusivity, molecular coupling and heat transfer.
Current applications in snowmaking and food science use trial and error approaches with fragments of ice bacteria. F-BioIce will provide the basis for a structure-based approach to enable a wave of new technologies that imitate the concepts of bacterial freezing. Such understanding will boost bioinspired innovations in cryopreservation of organs, cryo-therapy, food, cloud seeding and artificial icing and anti-icing surfaces. The same holds for atmospheric science: F-BioIce will provide key information to include the thermodynamics of biological ice nuclei in next generation climate models.
Experimental evidence of how INPs manipulate water is difficult to obtain. Very recent developments in laser-based ultrafast sum frequency generation (SFG) spectroscopy now allow us to probe molecular interfaces with high precision. We have had a strong focus on structural biology at interfaces for 10 years and within F-BioIce we are now applying new technologies to understand INP action. To solve the puzzle of biological ice nucleation, we will determine 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 froze so effectively. Freezing is highly dynamic and quick and we will therefore follow freezing at the surface of ice bacteria using ultrafast laser pulses. We will also follow how heat flows through the surface of the bacteria to understand whether INPs use a specific trick to funnel heat away rom the interface during freezing.
Current applications in snowmaking and food science use trial and error approaches with fragments of ice bacteria. F-BioIce will provide the basis for a structure-based approach to enable a wave of new technologies that imitate the concepts of bacterial freezing. Such understanding will boost bioinspired innovations in cryopreservation of organs, cryo-therapy, food, cloud seeding and artificial icing and anti-icing surfaces. The same holds for atmospheric science: F-BioIce will provide key information to include the thermodynamics of biological ice nuclei in next generation climate models.
Experimental evidence of how INPs manipulate water is difficult to obtain. Very recent developments in laser-based ultrafast sum frequency generation (SFG) spectroscopy now allow us to probe molecular interfaces with high precision. We have had a strong focus on structural biology at interfaces for 10 years and within F-BioIce we are now applying new technologies to understand INP action. To solve the puzzle of biological ice nucleation, we will determine 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 froze so effectively. Freezing is highly dynamic and quick and we will therefore follow freezing at the surface of ice bacteria using ultrafast laser pulses. We will also follow how heat flows through the surface of the bacteria to understand whether INPs use a specific trick to funnel heat away rom the interface during freezing.
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 are 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. The intense laser beams can be used to observe vibrations of molecules right from the interface. The vibrations report back on molecular structure such as water order, protein folding and side chain orientation.
We found that at room temperature the ice proteins were not interacting with the interfacial water to a great extend. 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.
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 are 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. The intense laser beams can be used to observe vibrations of molecules right from the interface. The vibrations report back on molecular structure such as water order, protein folding and side chain orientation.
We found that at room temperature the ice proteins were not interacting with the interfacial water to a great extend. 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.
Protein structure is the basis for the function of proteins. Its 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. Today, we have about 100.000 protein structures solved and stored in the Protein Data Bank (PDB). The structures have been solved using advanced methods such as X-ray crystalography, nuclear magnetic resonance and cryo-electron microscopy. However, when searching the database for protein structure for proteins bound to their native surfaces, there is not a single hit. The reason is that it is very challenging to probe protein structure at surfaces with the precision and reliability required for entry in the PDB. 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. We believe that this framework has the potential to fill the gap and provide information about interfacial proteins. 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, amyloridic 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.