Thesis
Creating an automated analysis toolbox for ultrafast 2D-IR spectroscopy
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2020
- Thesis identifier
- T15536
- Person Identifier (Local)
- 201567564
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Understanding protein structure is intrinsic to understanding their biological mechanisms. The technique of infrared (IR) spectroscopy can be used to analyse secondary structure of proteins using the amide I vibrational mode. However, the broad peak associated with this mode can be convoluted and extracting structural information can be difficult. The technique of two-dimensional infrared (2D-IR) spectroscopy can assist by spreading the spectral content across two frequency axes. It is a time-resolved method based on a series of sub-picosecond laser pulses that allows for ultrafast dynamics to be probed providing an advantage over more established structural analysis techniques, such as circular dichroism (CD) spectroscopy and X-ray crystallography.;The aims of this thesis are to assess the ability of 2D-IR spectroscopy to extract information pertaining to changes in structure of proteins in tandem with the multivariate analysis method of principal component analysis (PCA). This has been performed as a means to understand the spectral features of structure change in proteins upon perturbation and provides the basis for analysing protein dynamics using time-resolved infrared (TRIR) spectroscopy. As a testbed for this analysis, the previously well-characterised protein calmodulin (CaM) has been studied. CaM is a calcium binding messenger protein that is predominantly α-helical in structure. Ca2+ free CaM (apo) can bind up to four Ca2+ ions (holo) which produces a change in structure and α-helical content.;Thermal studies on both apo- and holo-CaM were performed and the quantitative results from 2D-IR spectroscopy produced good agreement with CD spectroscopy with a reduction in α-helix structure by 13% (CD) and 15% (2D-IR) observed for apo-CaM. Accurate differentiation between melting transitions and generic heating effects was achieved using the thermally stable holo-CaM as a reference.;A temperature-jump (T-jump) TRIR experiment was then established. The system was calibrated and determined to induce a 9 °C temperature rise in the sample. Comparison of non-equilibrium relaxations of apo-CaM and holo-CaM showed domain melting of apo-CaM begins on microsecond timescales with α-helix destabilisation. These observations enable the assignment of previously reported dynamics of CaM on hundreds of microsecond timescales to thermally activated melting, producing a complete mechanism for thermal unfolding of CaM.;CaM-drug binding studies were performed using IR and 2D-IR spectroscopy and small changes in structure and electrostatic properties were extracted utilising PCA. Separation into groups of ligand binding was achieved for IR spectroscopy with 2D-IR spectroscopy providing spectral evidence for the changes occurring.;The limitations of 2D-IR data processing for elucidating the small spectral changes and the developments made to ensure accurate extraction of spectral features are discussed.
- Advisor / supervisor
- Parker, Tony
- Hunt, Neil
- Baker, Matthew
- Resource Type
- DOI
- Date Created
- 2020
- Former identifier
- 9912881693102996
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