UBC Theses and Dissertations
Investigating biomolecular structure using fiber-optic UV resonance Raman spectroscopy Addison, Christopher James
The observation of biomolecular structure is critical for the fundamental understanding of biological function. In this work, Fiber-optic UV Resonance Raman Spectroscopy (FO-UVRRS) was employed to study a number of structure-function relationships. A suite of metal-containing dioxygenase enzymes were studied in order to determine the substrate protonation state in the enzyme-substrate complex. Two enzymes with a high degree of sequence identity were studied, where one naturally incorporates a Fe²⁺ metal and the other uses Mn²⁺. Both enzymes react with the same substrate to form an equivalent muconic semialdehyde product. The enzymes are capable of incorporating the non-native metal into the enzymes with no effect on the kinetic properties. The Raman results show that the nature of the metal has no effect on the substrate protonation state. The degradation of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) by BphD in the biphenyl catabolic pathway was studied using FO-UVRRS. Previous reports suggest that enol-keto tautomerization was a critical step in the mechanism of the carbon-carbon bond hydrolase. Hydrolytically impaired variants of BphD were used to study the binding mode of HOPDA. The results show that HOPDA binds to these variants in a strained-enolate geometry and does not undergo an enol-keto tautomerization upon enzyme binding. A fundamental FO-UVRRS study of locked nucleic acid (LNA) olgiomers was performed. LNA bases contain a C4’ to O2’ methylene bridge within the furanose ring of the nucleotide. The research results show that incorporation of a LNA induces a conformational change in the glycosyl bond between the backbone and the base. The results also show that incorporation of an LNA base induces changes in the secondary structure of the nucleic acid. In another study, chemical contamination of synthetic DNA oligomers was observed using FO-UVRRS. The data showed that commercially-available oligomers purified using standard desalting conditions are contaminated with residual benzamide. The results show that a previously assigned –NH₂ scissors vibration in the Raman study of oligomers overlaps with a prominent benzamide vibrational band, calling into question these previous assignments. The results also demonstrate that the extent of benzamide contamination varies from sample-to-sample and between different commercial sources.
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