Studying biomolecules using photoactivation and mass spectrometry techniques

Abstract

The ideal experiment for the understanding of structural biology will yield information on both structure and function in a single experiment. Standard approaches for the investigation of biomolecules usually includes NMR and X-Ray Crystallography, these come with limitations including complex spectra from NMR and in many cases proteins will not crystallise where they contain intrinsically disordered regions. Other methods include UV/Vis and IR investigations which will give information on the secondary structure but are limited when it comes to the understanding of the quaternary structure. Ion mobility mass spectrometry (IMMS) has increasingly been utilised to investigate protein structure and overcome some of these issues. IMMS provides both stoichiometric and structural data that can be used to probe the stability of global fold and the non-covalent interactions within a given protein complex. The data produced is also far less complex than results from NMR making it an easy process to apply and can be applied to a range of biomolecules with minimal changes to the settings. This thesis describes the use of IMMS to interrogate the functional fold of proteins via a modified Synapt G2-S that incorporates light in the form of high powered pulsed UV lasers as well as LED. The UV lasers have been implemented to allow photodissociation of proteins (Ultraviolet Photodissociation, UVPD) either pre- and post-ion mobility. These arrangements allow us to link the fragments to the protein fold and hence gain a better understanding of the perturbation of protein structure. On the same IMMS instrument, a rig was constructed to permit LED illumination of analytes within the electrospray (ESI) tip, and to then use the mass spectrometer to report on the results of such in solution photoactivation. This is applied to the investigation of photoactive proteins and a methodology has been developed to perform this work under minimal light activation. UVPD-IMMS investigation of well-studied monomeric proteins produces distinctive fragments that can be related to the non-covalent interactions which determine the global fold. UVPD-IMMS experimental approach was extended to large multimeric proteins, where further information of the native fold could be determined. Alongside UVPD-IMMS, collision induced dissociation (CID) IMMS measurements were also performed on well-studied monomeric proteins, distinctive fragments could relate to the precursor charge state. The use of multiple fragmentation strategies produces complex rich datasets and these were compared using PLS-DA to produce scores plots. From these comparisons were made between conformationally different proteins fragmented via UVPD and between different charge states fragmented via CID. We observed that the conformations and charge states yielded significantly different fragmentation patterns that could be separated via PLS-DA and give the option to get further information than the standard fragment assignments. The use of LEDs to activate proteins is described in a protocol aimed at those with general knowledge of MS but unaware of the added benefit of applying this to photoreceptor proteins. We describe the procedure for the construction of the apparatus, show exemplar data and acquisition settings that have been developed on a range of different mass spectrometers. Exemplar data for photoreceptor proteins and a commercially available small molecule is also shown for the set-up. The photoreceptor proteins chosen cover a range of the UV-visible spectrum to show the flexibility of the LED set-up. Finally, we have utilised the improved capabilities to work with photoreceptors and light-reactive proteins to investigate how the tetramer formation of WT TtCarH when in the presence of Adenosylcobalamin. This was performed under minimal photoactivation and required special care when handling. The step-wise potential mechanism was determined through a combination of MS, stop-flow and molecular dyna

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Perdita Barran (Supervisor)