Please use this identifier to cite or link to this item: http://doi.org/10.25358/openscience-6847
Authors: Lukas, Max
Title: Toward Understanding Bacterial Ice Nucleation - Interface-specific nonlinear spectroscopy used to unravel the working mechanisms of bacterial ice nucleation proteins
Online publication date: 12-Apr-2022
Language: german
Abstract: Small droplets of pure water, as they are found in our atmosphere, can be supercooled to temperatures close to -40 °C. Above this temperature, the formation of a stable initial ice nucleus can be triggered by interaction with aerosol particles. The properties that make a good ice nucleator (IN) have been under discussion for decades, based on the idea that the underlying mechanism must involve ordering of adjacent water molecules into an ice-like structure. A variety of efficient INs have been identified, of which the bacterium Pseudomonas syringae exhibits exceptional ice nucleation efficiency, facilitating freezing at ~ -2 °C. Although it is known that specialized ice nucleation proteins (INPs), anchored to its cell membrane, are responsible for the nucleation mechanism, this same membrane integration impedes the analysis of the protein's three-dimensional structure and hence clarity about its working mechanism. In the first chapter of this thesis, the previous fundamental findings concerning bacterial ice nucleation proteins, in specific of P. syringae, are compiled and discussed. These indicate that a distinctive feature of the bacterial INs, the precise assembly of the INPs into functional aggregates, is crucial for their efficiency. It has been shown that this aggregation largely depends on environmental conditions; the reasons, however, have remained unclear. Therefore, we conduct studies on the influences of pH as well as ion-specific interactions. In this thesis, we use interface-specific sum-frequency generation (SFG) vibrational spectroscopy to investigate the molecular-level interaction of the proteins with water. As a tool for evaluating the effects on the functional level, we utilize the Twin-plate Ice Nucleation Assay (TINA). This droplet freezing experiment yields so-called freezing spectra, which give information on the INs present in the sample, active at distinct freezing temperatures. Applying this combined approach, we discover that the absence of the natural net charge at the isoelectric point of the INPs is accompanied by the complete loss of the most efficient Class of functionally aggregated INs, which is an important detail of this puzzling class-selective pH dependence (Chapter 4). Furthermore, we link the effects of ions on the ice nucleation efficiency to their specific interactions with the protein surface, by studying INP solutions in the presence different salts (Chapter 5). We find that these specific ion effects on the threshold freezing temperatures follow the Hofmeister series and propose two competing explanatory approaches based on molecular dynamics simulations. Next, we make use of ice-affinity purification methods and focus on investigations of temperature effects on the protein structure (Chapter 6). Using circular dichroism and Amide I SFG spectroscopy, we demonstrate that its three-dimensional structure is not altered at low temperatures but drastically changed upon heat treatment, which in turn results in the loss of its ice nucleation activity. Moreover, we show that a previously reported increased order of water in contact with INPs at low temperatures is also found for such heat-inactivated INP solutions, and therefore does not constitute a sufficient condition for ice nucleation activity. Finally, we conclude our studies and formulate a perspective on the research of bacterial INPs (Chapter 7). We emphasize the importance of functional aggregation and environmental conditions and hence consideration of the crucial role of the bacterial membrane. An understanding of the outstanding ice nucleation efficiency of bacterial INPs demands an interdisciplinary approach to link functional-level effects observed with high-throughput freezing assays with changes of the proteins' conformation and their interactions with water. In addition to the introduction into the fundamentals of nonlinear optics and conventional SFG spectroscopy, as used in the presented studies, this thesis also contains a detailed discussion of phase-resolved SFG (PR-SFG). Capturing the phase of SFG signals has several advantages; most prominently, it provides direct information on the molecular orientations at the probed interface. In Chapter 3, a novel setup is presented, which overcomes the inherent technical challenges in a way that future studies, not only of ice-nucleating bacteria as proposed in as Chapter 8, can benefit from these advantages without significant additional experimental effort.
DDC: 500 Naturwissenschaften
500 Natural sciences and mathematics
540 Chemie
540 Chemistry and allied sciences
570 Biowissenschaften
570 Life sciences
Institution: Johannes Gutenberg-Universität Mainz
Department: MaxPlanck GraduateCenter
Externe Einrichtungen
Place: Mainz
ROR: https://ror.org/023b0x485
DOI: http://doi.org/10.25358/openscience-6847
URN: urn:nbn:de:hebis:77-openscience-f0b32aaf-fa33-48b3-895c-bcfa037b78d98
Version: Original work
Publication type: Dissertation
License: In Copyright
Information on rights of use: http://rightsstatements.org/vocab/InC/1.0/
Extent: 144 Seiten, Diagramme, Illustrationen
Appears in collections:JGU-Publikationen

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