Analysis of Non–Covalent Systems through Mass Spectrometry Techniques

Abstract

Non–covalent interactions represent a major area of interest across the scientific community, with the interactions of biomolecules being an interesting and influential field. Regulating protein function by influencing binding of co–factors and other proteins is at the forefront of many research directions. However, non–covalent complexes can present challenging targets for analysis as many techniques are unable to differentiate between species in a heterogeneous population. Mass spectrometry (MS) experiments provide the unique ability to isolate individual components in a mixture prior to analysis, even when they exist in a dynamic equilibrium. When this is combined with hybrid techniques such as ion mobility–MS, along with fragmentation, the differences incorporated within a complex can be identified structurally and related back to the binding interface. The work presented in this thesis demonstrates how MS and related techniques are suitable methods to address the challenge of analysing non–covalent complexes The first system in chapter two is a DNA aptamer that has been designed to bind an antibiotic kanamycin. Aptamers represent an emerging field in biopharmaceuticals due to their ability to provide specific antigen binding analogous to antibodies whilst providing advantages in chemical synthesis and immune response. The interaction between these two molecules was assessed using a variety of MS techniques to elucidate the nature of the interface, if the structure of the molecule is pre–organised for binding, and the conformational changes that binding induced. Additional sequence constructs were compared for their binding and structural similarities. The systems under scrutiny in chapter three belong to a class of anion sensing molecules designed to detect pyrophosphate via displacement of a dye molecule, with a high specificity over similar cationic molecules. These unique sensors have the potential to monitor cellular respiration by detecting pyrophosphate released from ATP. The sensors consist of a circular peptide scaffold with two flexible arms each leading to bound Zn (II) ions, with a variety of permutations of length and size examined. Gas–phase kinetic studies were performed to assess efficacy and selectivity of binding pyrophosphate. Subsequently, ion mobility and computational data were used to determine structure–function relationships. Chapters four and five consider the structural properties of the transcription factor c–MYC and its binding partner MAX. Deregulation of c–MYC is implicated in human cancer progression, therefore making it an attractive pharmaceutical target. However, the leucine zipper binding region of the two proteins is intrinsically disordered, undergoing a disorder to order transition upon binding which presents difficulties for characterisation using traditional structural techniques. These chapters explore the effects of a series of small molecules that have been shown to inhibit dimerisation of c–MYC and MAX through a postulated stabilisation of these disordered states. Peptides derived from the proteins are investigated in the work presented in chapter four, whilst in chapter five extended sequences are interrogated. Further work in chapter five also considers the ability of the dimers to bind DNA in the presence and absence of ligands.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Richard Winpenny (Supervisor) & Perdita Barran (Supervisor)