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Biophysical studies of cytochrome P450 enzymes - biomimetic membranes as platforms to study protein-protein interactions
thesisposted on 22.03.2017, 01:20 by Praporski, Slavica
The structure of a cellular membrane is a complex amalgamation of the phospholipid bilayer and proteins that reside in it; i.e. ion-channels, enzymes, G protein-coupled receptors, to name a few. The role of the membrane in cellular function and homeostasis has been long recognised; however, studies of proteins that reside in it are inconvenienced by the lack of enough purified native protein to allow classical crystallographic and spectroscopic examination. The advent of surface-based techniques that require only a small amount of protein and allow biomimetic membrane deposition, provide new avenues to study these proteins, their interactions with the membrane and protein-protein interaction that occur in the membrane. This thesis employs the quartz crystal microbalance with dissipation monitoring (QCM-D) technique to study peptide and protein interactions with the biomimetic membranes. QCM-D provides the suitable surface that can be modified with the membrane and can record changes over time that occur upon protein or peptide binding. Furthermore, QCM-D can monitor rearrangements and fluctuations in the viscoelastic properties of the resulting composite layers, which is not possible to do with any other technique. This technique enabled a study of the mechanisms of membrane disruption by lytic antimicrobial peptides, needed for novel drugs to treat multi-resistant bacterial infections. Also, the protein-protein interactions which are important in the function of steroidogenic enzymes residing in the endoplasmic reticulum membrane were investigated. Prior to the study of peptides and proteins interacting with the biomimetic membranes, it was first necessary to characterise the deposition processes of the biomimetic membrane on the gold coated QCM sensor surface. Deposition and membrane formation from liposome solutions onto the gold-coated QCM sensor surface, that was first modified with the self-assembled monolayer (SAM) of mercaptopropionic acid, was investigated, and the results of these studies are presented in Chapter 2 (Paper 1). Once the membrane deposition was developed as a reproducible protocol, the interaction of a family of antimicrobial peptides was studied with different biomimetic membranes, and the results of these experiments are presented in Chapter 3 (Papers 2 and 3). These antimicrobial peptides were chosen for initial studies of membrane disruption in order to better understand and optimise the QCM-D technique before attempting to investigate interactions of proteins with the biomimetic membranes. Additionally, three more papers, that are not included in the main thesis body, also involve experiments of this kind that were undertaken during this project, and are included in the appendices. Development of a methodology to investigate protein-protein interactions using QCM-D necessitated the investigation of a single protein adsorption onto the gold-coated QCM chips modified with octanethiol-SAM. This was followed by adsorption studies of more complex system involving a bacterial cytochrome P450 enzyme and its partner proteins on various SAMs. This study was able to answer important questions about the surface interactions of these proteins. Results of these studies are presented in Chapter 4 (Paper 4 and section 4.2). Ultimately, Chapter 5 describes the investigation of several steroidogenic cytochrome P450 enzymes, their redox partner, and accessory proteins interacting with a range of biomimetic membranes. Paper 5 describes the results of a study dealing with the phenomenon of protein homodimerization in vivo and in vitro. Paper 6 describes the results of a study where the activities in biomimetic membrane of the two P450 aromatase enzymes were compared and the effect of the redox partner on each of the enzymes assessed. Papers 7 and 8 describe the results of a study on P450c17 enzyme system (including cytochrome P450 oxido-reductase (CPR), cytochrome b5 and a cytochrome b5 mutant). Paper 7 represents a PART 1 of this study in which the basic questions were considered, i.e. how these proteins bind to the biomimetic membrane individually and in sequence, and how their presence affects the properties of the membrane. Also, it describes the protein-protein interactions that occur during binding, highlighting their specificity. The activity of P450c17 was also tested, while the enzyme was bound to the biomimetic membrane, and positive results lead to a further exploration of this system. The results of this next stage of experiments are presented in Paper 8, representing a PART 2 of the study. Here, the effects of the CPR and the cytochrome b5 proteins on the activity of P450c17 were investigated, and the results highlight the role of protein-protein interactions mediated by the biomimetic membrane.