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.
