Applications of electron paramagnetic resonance in biomedicine

Abstract

Spectroscopy and imaging are widely used to characterise systems structurally and functionally, and this information allows for the rational development of novel drugs and theranostics in many diverse areas of medicine. This thesis focuses on applying Electron Paramagnetic Resonance (EPR) techniques to determine the structure and function of protein molecules. A feasibility study to develop paramagnetic probes for EPR imaging is also presented.In EPR the probe is a paramagnetic centre and information is obtained by measuring interactions of this probe with its environment. In this thesis continuous wave (CW) EPR and Double Electron Electron Resonance (DEER) in conjunction with a site-directed spin labelling (SDSL) were employed to study structure and function of spin-labelled protein molecules. The techniques allow examination of molecular systems that cannot be crystallised or are too big for efficient NMR investigation. DEER provides information on distance distributions between two spin labels located on Cys-mutated residues of the protein molecule and can access longer distances in comparison with NMR. CW EPR provides information about the mobility of a single spin label which can be used to characterise protein dynamics. Structural modelling was used to combine the crystal structures and Molecular Dynamics (MD) simulations with DEER distance constraints to present a DEER-based structural model of the conformational variability of the protein molecule.Chapters 2 and 3 focus on characterising two metal ion substrate binding proteins (SBP), Zn2+-binding AdcA and Mn2+-binding PsaA, using DEER and CW EPR. The ATP-Binding Cassette (ABC) permeases, with which the SBPs are associated, are a primary importer used by bacteria to scavenge the essential first-row metal ions (e.g. iron, zinc, manganese) from a host environment. The process is essential for bacterial survival and propagation. Understanding the metal ion acquisition mechanisms by these SBPs can provide new opportunities for targeted drug development. Collectively, the CW EPR and DEER data along with crystal structures, differential scanning fluorimetry (DSF), Molecular Dynamics simulation, and smFRET microscopy, were able to determine a structural model for Zn2+-binding in the AdcA protein referred to as “trap-door” mechanism. Our data show that the “trap-door” mechanism employed by AdcA is different from the “spring-hammer” mechanism employed by PsaA.Non-Ribosomal Peptide Synthetase (NRPS) are the topic of chapter 4. NRPS’s are a family of mega enzymes that produce a diverse range of pharmaceuticals, yet how these molecular machines operate to produce such complex chemicals is very poorly understood. This project will investigate module 7 of the teicoplanin NRPS, a 200 kDa protein, which is the last step in the production of the glycopeptide antibiotics (GPA). A comprehensive structural and functional understanding of the teicoplanin NRPS machinery will provide a paradigm for this enzyme family, enabling the tailored re-engineering of the in vivo NRPS biosynthesis for the development of new antibiotics and pharmaceuticals. This thesis concentrates on a di-domain complex PCP-X from module 7 (Tcp12) of teicoplanin NRPS which contains the peptidyl carrier protein (PCP) domain and the X-domain. These two domains are joined by a long linker which is thought to be flexible. The goal is to investigate conformational dynamics of the PCP-X construct in different states. This includes the PCP domain in loaded and unloaded states, and how the PCP-X construct interacts with an OxyB (P450) protein. Using DEER data from a large number of double spin-labelled PCP-X mutants, rigid-body structural models based on the domain’s crystal structures were investigated to characterise the mobility of the PCP-X di-domain and compare it to very limited crystal structure data of the larger domain construct.Chapter 5 describes the application of the Maximum Entropy (MaxEnt) approach to 2D HYSCORE image reconstruction and its comparison with the conventionally utilised Discrete Fourier Transformation (DFT). HYSCORE is a widely used electron spin echo envelope (ESEEM) experiment used for measuring the nuclear quadrupole and hyperfine couplings. While EPR experiments are generally very sensitive, the time required for two and higher dimensional techniques are a significant limitation. The results show that the MaxEnt algorithm allows sampling of data non-uniformity, provides better sensitivity and an increase in resolution compared to DFT.Lastly, chapter 6 describes the development of PEG-based hyperbranched polymers and pPEGMA-coated gold nanoparticles as EPR imaging agents and sensors. Both nanoparticles have been shown as promising platforms that can be functionalized with targeting ligands and various imaging probes (i.e. a paramagnetic spin label) for the purpose of using multiple molecular imaging modalities for in vivo theranostics. The choice of a spin label dictates the possible application of the final agent, either for EPR imaging or EPR spectroscopy. The preliminary study assessed the sensitivity of commercially available EPR equipment and showed that nitroxides are more suitable for use as a sensor rather than an imaging probe. Future work based on the results in the thesis are discussed at the end of this chapter.