Creating an automated analysis toolbox for ultrafast 2D-IR spectroscopy

Student thesis: Doctoral Thesis

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.
Date of Award16 Apr 2020
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorNeil Hunt (Supervisor) & Matthew Baker (Supervisor)

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