Tiemessen, David
(2021)
Studying the intra and intermolecular processes of biomolecules using two dimensional infrared spectroscopy.
PhD thesis, University of Nottingham.
Abstract
This Thesis focuses on using ultrafast time-resolved infrared spectroscopy and two-dimensional infrared spectroscopy (2D-IR) for determining the structure and dynamics of a system.
Chapter 1 gives an introduction to 2D-IR spectroscopy which is a technique that involves vibrationally exciting a sample, with an initial IRpump pulse, then after a waiting time the sample is probed using an IRprobe pulse. A spectrum is obtained by scanning the frequency of the IRpump pulse at a fixed waiting time. This technique allows the multiple transitions to be probed and valuable information (e.g. the coupling of vibrational modes) such as how energy is transferred, both within a molecule and to its environment, to be elucidated.
Chapter 2 describes investigations of the photodissociation of Fe(CO)5. This was chosen as a model system to investigate controlling the process of photodissociation using a vibrational excitation in solution. Transient 2D-IR spectroscopy (T-2D-IR) is a powerful tool, as electronic and vibrational pump pulses can be combined together with a sensitive IR probe to interrogate such processes on the ultrafast timescale. The T-2D-IR spectra of Fe(CO)5 in heptane and CH2Cl2 are presented and we demonstrate that photolysis of Fe(CO)5 can be controlled using a vibrational excitation such that the yield of the photoproducts can be altered. We find that for Fe(CO)5 in heptane, exciting either vibrational mode 2 ps before photolysis by a 266 nm UV pulse results in a small increase in the formation of Fe(CO)3. Interestingly for CH2Cl2, exciting only the lower vibrational mode 2 ps before photolysis by a 266 nm UV pulse results in decrease in the formation Fe(CO)3, no change is observed if the higher vibrational mode is excited. No change in the formation of Fe(CO)3 was also observed when the sample was immediately excited with an IRpump pulse after the UVpump pulse. These observations are discussed and the process of vibrationally controlling the photodissociation of Fe(CO)5 is clearly a very complex problem which requires further work to understand the underlying mechanism.
Chapter 3 discusses the use of 2D-IR spectroscopy to investigate the gelation mechanism of carrageenan. Polysaccharide gels are a very important component in the food, pharmaceutical and cosmetic industries. In the food industry these gels act as thickeners and stabilisers. They help give products their structure and physical properties such as texture. Carrageenan is one such polysaccharide gel and contains organosulfate groups. It has been shown that it limits ice crystals growth in frozen foods which is crucial to the stability of the product. There are three different types: ι, κ and λ-carrageenan, each of these differ in the number and position of the organosulfate functional groups. The interactions in and around these functional groups are thought to be critical to the properties of the gel and the gelation mechanism. 2D-IR spectroscopy was used to directly probe these functional groups and how their interactions change as gelation occurs. For ι-carrageenan in the solution state, the organosulfate groups exist in a large broad range of environments. Upon gelation, cross peaks were observed in the 2D-IR spectra, indicating that the organosulfate groups exist in three main conformations which all interact with each other. The different environments are thought to relate to different cation interactions. These distinct environments were not observed for κ-carrageenan. We found that the spectral slices remained similar at different temperatures and this was interpreted to be due to the structural differences between ι- carrageenan and κ-carrageenan, with the latter only having one organosulfate group per monomer. The solvent dynamics of carrageenan was also investigated using ferrocyanide as a probe molecule by measuring the spectral diffusion using 2D-IR. The energy of water H-bonds in carrageenan was determined to be approximately half that of bulk water. This was thought to be because of disrupted H-bonding networks. While the bond reformation time was estimated to be approximately four times that of bulk water and was assumed to be due to confined water bonding networks. There was no discernible difference between the two types of carrageenan.
Chapter 4 investigated the interaction between salivary mucins and tea polyphenols. 2D-IR spectroscopy was used to probe the interactions of the milk protein, β -lactoglobulin, and the green tea polyphenol, epigallocatechin gallate, specifically using the amide I band in order to observe any conformational changes of the protein. The N-terminal domain of MUC5B (NT5B) has been previously shown to interact the most with theaflavins, specifically theaflavin digallate. Again, using 2D-IR spectroscopy, the secondary structure of NT5B was determined to be formed of mainly β-sheet and some α-helix. When NT5B and theaflavin digallate interacted, conformational changes were observed. A reduction in β-sheet was observed while the α-helix conformation remained largely unchanged. There was also an indication of formation of aggregates and the results were interpreted to indicate that the β -sheet conformation is potentially important in the process of mucins cross linking.
Chapter 5 outlines the details of the equipment and experimental approaches used in the Thesis, and two appendices contain other work I have been associated with during my PhD. Appendix B contains the investigation of tryptophan to heme electron transfer in myoglobin, using TRIR spectroscopy where the experimental data had been previously collected with my work undertaking all the analysis and interpretation of these results. Appendix C details an investigation of lignin formation, which is a complex biopolymer, and is important in the formation and function of cell walls. Confocal Raman microscopy was used to show chemically distinct lignin was produced when altering a cell signalling pathway, where, again the experimental Raman data had been previously collected with my work undertaking the multivariate analysis and interpretation of these results.
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