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Fourier transform infrared spectroscopic characterisation of biomolecules

Jackson, Michael; (1990) Fourier transform infrared spectroscopic characterisation of biomolecules. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Fourier transform infrared (FTIR) spectroscopy has been applied to the characterisation of structural changes in a range in biological molecules. The polymorphic phase behaviour of non-hydroxy fatty acid galactocerebrosides was shown to be the result of extensive rearrangements of the headgroup hydrogen bonding network. Formation of a metastable cerebroside form upon rapid cooling was shown to be due to dehydration of the lipid. Polymorphism was abolished by cholesterol and DPPC at levels normally found in myelin. The temperature dependence of the secondary structure of a range of proteins was investigated. It was demonstrated that several predominantly α-helical proteins partialy unfolded at 70-80°C forming intermolecular β-sheet structures. This behaviour was demonstrated for globular, fibrous and membrane proteins. Addition of Ca++ to α-helical Ca++-binding proteins prevented unfolding of the helices. Calmodulin, parvalbumin and troponin C were shown to exhibit FTIR spectra in H2O characteristic of helical/disordered proteins. Deuteration resulted in significant shifts of the amide absorption to wavelengths consistent with disorganised structures, in contrast to CD spectroscopic studies which suggested a helical structure. Disruption of protein-solvent interactions with glycerol increased the frequency of the amide I band for parvalbumin. It was suggested that the unusual frequency of the amide I band in 2H2O is related to the high degree of protein- solvent interaction in these proteins conferred by their open structures. Examination of the FTIR spectra of the Ca++-ATPase of sarcoplasmic reticulum under conditions known to stabilise the protein in a variety of conformational substates indicated no significant changes in secondary structure during the catalytic cycle. This result was confirmed by curve fitting analysis of the FTIR data. A number of flaws became apparent in this analysis, the most important being the assumption that all secondary structures have equal molar absorptivities. Studies on polylysine showed this not to be so.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Fourier transform infrared spectroscopic characterisation of biomolecules
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10116691
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