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Title: Structural and biophysical characterization of photoswitchable peptides and their complexes
Author: Wysoczanski, Piotr
ISNI:       0000 0004 2735 6242
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2012
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Networks of biomacromolecular interaction form the core of internal cellular logic where a wide range of input signals get analysed and results in a genetically programmed output. This description holds not only for healthy cells but is true also for those virally infected when a virus implements new biomacromolecular elements and modifies existing networks. Those hostile interactions are crucial for the HIV virus replication cycle. Due to the scale-free nature of biomacromolecular interaction networks they are prone to malfunction if a hub (node with the highest interactions) is damaged. Perhaps the best example of such a situation can be observed in the apoptotic machinery. Overexpression, deletion or mutations (depending on the context) in apoptotic proteins can lead to various diseases, most prominently cancer. Therefore current research efforts concentrate on designing inhibitors of malfunctioning interactions. Occasionally interaction between the two binding partners relies on a well-defined secondary structures element. This allows to engineer this element and use it to bias the given interaction equilibrium towards dissociation. One such approach concentrates on α-helices that are crosslinked with azobenzene derivatives. This permits not only to increase the helical content of a peptide, which translates to increased affinity (potency), but transplants a photo-controllability. Photoswitchable peptides created in this way can be controlled externally; their affinity towards targets can be effectively photomodulated. The first chapter of the thesis concentrates on the development and evaluation of photocontrollable peptides derived from the arginine rich region of the HIV type I Rev protein. Rev, which is an early product generated from the HIV genome, binds to RRE containing mRNA and removes it from the nucleus, allowing expression of unspliced or singly spliced mRNA into different protein products. As a consequence the RRE-Rev interaction controls the infection cycle of the HIV type 1, as these later products are necessary for virus particle assembly. The α-helical peptide derived from Rev was shown to retain most of the affinity to RRE. Rev-RRE serves as a prototypical protein-RNA interaction; a model for the development of the first photo-controllable peptide that targets RNA. This research presents two approaches for the peptide production: chemical synthesis and biosynthesis (overexpression of the Rev peptide gene). Initially the water soluble azobenzene derivative was used bearing two negatively charged sulphonate substituents on the aromatic rings. Surprisingly, the first generation of the peptides had substantially decreased affinity to RRE VI as compared to the wild type Rev peptide. By applying computational chemistry techniques a possible rationalisation was found: sulphonates interact strongly with crucial arginines on the peptide making them unavailable to engage the phosphate backbone; additionally the peptide was forced into non-helical conformation prior to binding. This hypothesis was validated by crosslinking Rev peptides with azobenzene derivative deprived of the sulphonates. The second generation of Rev peptides binds RRE with high affinity and the interaction in question can be controlled effectively by light. The second chapter of the thesis focuses on the structural characterization of a photocontrollable Bak peptide bound to Bcl-xL – a prominent member of the Bcl-2 family of proteins. The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. In this thesis the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-xL is reported. This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms, is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry