Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Airborne microorganisms utilize these proteins to enhance their survival by reducing their atmospheric residence times. INpro also have critical environmental effects including impacts on the atmospheric water cycle, through their role in cloud and precipitation formation, as well as frost damage on crops. INpro are ubiquitously present in the atmosphere where they are emitted from diverse terrestrial and marine environments. Even though bacterial genes encoding INpro have been discovered and sequenced decades ago, the details of how the INpro molecular structure and oligomerization foster their unique ice-nucleation activity remain elusive. Using machine-learning based software AlphaFold 2 and trRosetta, we obtained and analysed the first ab initio structural models of full length and truncated versions of bacterial INpro. The modeling revealed a novel beta-helix structure of the INpro central repeat domain responsible for ice nucleation activity. This domain consists of repeated stacks of two beta strands connected by two sharp turns. One beta-strand is decorated with a TxT amino acid sequence motif and the other strand has an SxL[T/I] motif. The core formed between the stacked beta helix-pairs is unusually polar and very distinct from previous INpro models. Using synchrotron radiation circular dichroism, we validated the β-strand content of the central repeat domain in the model. Combining the structural model with functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. Using computational docking of the new INpro model based on rigid-body algorithms we could reproduce a previously proposed homodimer structure of the INpro CRD with an interface along a highly conserved tyrosine ladder and show that the dimer model agrees with our functional data. The parallel dimer structure creates a surface where the TxT motif of one monomer aligns with the SxL[T/I] motif of the other monomer widening the surface that interacts with water molecules and therefore enhancing the ice nucleation activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity.
B29Nε-lithocholyl-γ-l-βGlu-desB30 human insulin [NN344] belongs to a group of insulins with fatty acid or sterol modifications. These insulin analogues have been found to form subcutaneous depots upon injection and hereby have a protracted release profile in vivo. In the present study, B29Nε-lithocholyl-γ-l-Glu-desB30 human insulin was investigated using in-solution small-angle X-ray scattering (SAXS) at chemical conditions designed to mimic three stages during treatment in vivo: in-vial/pen, postinjection, and longer times after injection. We found that the specific insulin analogue formed a mixture of mono- and dihexamers under in-vial/pen conditions of low salt and stabilizing phenol. At postinjection, conditions mimicking a subcutaneous depot, B29Nε-lithocholyl-γ-l-Glu-desB30 human insulin, formed very long straight soluble hexamer-based rods stacked along the Zn(II)-axis. The self-assembly was triggered by an increase in salt concentration when going from vial to physiological conditions. Mimicking longer times after injection and the additional removal of phenol caused the length of the rods to decrease significantly. Finally, we found that the self-assembly could be controlled by varying the amount of modification at the interaction interface by making mixed hexamers of B29Nε-lithocholyl-γ-l-Glu-desB30 and desB30 human insulin. This opens extra possibilities for controlling the release profile of very-long-acting insulins.
Ice active bacteria can catalyze water freezing at high subzero temperatures using ice nucleating proteins (INPs) located at their outer cell walls. INPs are the most effective ice nucleators known and are of significant interest for agriculture, climate research, and freeze/antifreeze technologies. The aggregation of INPs into large ice nucleation sites is a key step for effective ice nucleation. It has been proposed that ice active bacteria can drive the aggregation of INPs and thereby trigger ice nucleation. However, the mechanism of INP aggregate assembly and the molecular processes behind the activation are still unclear. Both biochemical pathways and activation through electrostatics have been proposed based on experiments with lysed ice active bacteria. For a more direct view on the assembly of INPs, we follow the structure and water interactions of a synthetic model INP of the well-studied ice bacterium Pseudomonas syringae at the air–water interface as a function of the subphase pH. By combining sum frequency generation spectroscopy with two-dimensional infrared spectra, we conclude that self-assembly and electrostatic interactions drive the formation of ordered INP structures capable of aligning interfacial water.
Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Their critical role in rain formation and frost damage of crops together with their diverse commercial applications warrant an in-depth understanding of their inherent ice nucleation mechanism. We used the machine-learning based software AlphaFold to develop the first ab initio structural model of a bacterial INpro which is a novel beta-helix structure consisting of repeated stacks of two beta strands connected by two sharp turns. Using the synchrotron radiation circular dichroism, we validated the β-strand content of the model. Combining functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity and the basis for investigating the mechanistic role of INpro-induced ice formation in the atmosphere, and for commercial design and production of ice-nucleating particles for industrial applications.
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