Instalación y Configuración del Sistema Gestor de Base de Datos

Biochemical studies such as protein folding often rely on repeating experiments multiple times in order to generate reliable and statistically valid data. Consequently, sample consumption can be high, driving the need for efficient and reliable material supply method.

Initial studies of prion proteins relied on material purified from hamster brain extracts – a method both time consuming and generating low yields. A suitable illustration of the issue is a fact that one of the most efficient protocols based on immobilised Cu2+ and cation exchange chromatography of detergent-extracted material provided yields in range of only 120 μg per 100 g of brain tissue (Pan et al., 1992). Thus, structural studies of PrP benefitted greatly from development of bacterial overexpression systems. These recombinant expression systems provided an effective method of generating large yields in a much simpler manner, consuming less materials and labour.

Recombinant PrP from a number of species obtained from bacterial expression allowed for high resolution structural analysis of prions through NMR spectroscopy

(Hornemann et al. 1996; Viles et al. 1997; Zahn et al. 2000). This, coupled with determination that the structure of native PrP purified from brain tissue is identical to recombinant one (Hornemann et al. 2004) allowed for recombinant prion protein to be widely used as a model of PrPC behaviour.

Studies presented in this thesis focus on investigating folding pathways of prion proteins from different species via two primary methods: the study of early folding kinetics of mouse and Syrian hamster prion protein in order to investigate the influence of a disease preventing mutation Q167R on the folding pathway of PrP, and fibrilisation studies of hamster, mouse and elk PrP to determine the effects of disease-related

mutations on formation of fibrils.

The early folding pathway of prion proteins was investigated using a number of spectroscopic and kinetic techniques such as circular dichroism and tryptophan fluorescence.

42

Tryptophan fluorescence measurements are particularly useful for studying protein folding pathways in aqueous solutions, since tryptophan residues within investigated protein can be selectively excited and act as a highly sensitive fluorescent probes responding to changes in their local environment induced by folding or unfolding of polypeptide chains. Upon unfolding of the protein, the tryptophan residues buried in hydrophobic core of the structure become solvent-exposed which is reflected in their spectral properties.

Application of tryptophan fluorescence to structural studies of PrP required carefully modified constructs to be used. The structure of prion proteins employed in

fluorescence studies encompasses residues 90–231 of Syrian hamster and 91–230 of mouse proteins, representing the globular folded domain and short unstructured N- terminus section of PrPC as well as proteinase K-resistant core of PrPSc. The remainder of the N-terminus containing residues 23–90 of MoPrP and 23–89 of SHaPrP is

unstructured and thus unlikely to have any influence on the folding of globular domain. The two native tryptophan residues present in globular domain of PrP are solvent- exposed even in the folded state and thus unsuitable for protein folding monitoring by fluorescence. Consequently, both of the residues had to be removed by introducing point-mutations which replaced them with phenylalanines. Similarly, six tryptophans present in the unstructured domain would have to be removed if the N-terminus part of the protein was to be retained, a process both time and resource consuming. Since that particular region was deemed to be unimportant for folding of globular domain and consequently removed, only the residues in the globular domain were replaced.

Following the removal of any undesirable native residues, site-directed mutagenesis can be used to introduce tryptophan residues in strategically important positions, allowing them to act as fluorescent reporters of structural changes. Such a strategy has been successfully employed by members of author‟s research group in kinetic and

equilibrium folding studies of prion proteins (Sanghera and Pinheiro 2002, Kazlauskaite

et al. 2003, Jenkins et al. 2008, Robinson and Pinheiro 2009).

Fibril formation by mouse, Syrian hamster and elk PrP was investigated in a series of fibrilisation and oligomeristaion studies using ThT fluorescence (Gill et al.

2009) involving both truncated and full length constructs. Positively charged ThT is known to bind to negatively charged nucleic acids as well as fibrils formed by α- synucleins and prion proteins. Upon binding, ThT molecules which exhibit low fluorescence emission upon excitation at 450 nm in water, show several-fold

43

enhancement in fluorescence intensity. This increase in emission is characteristic only for bound ThT and therefore is often used as a marker of fibril formation. Since ThT fluorescence measurements utilize different excitation and emission wavelengths than tryptophan fluorescence, both native tryptophans and unstructured N-terminus domain of PrP could be retained to investigate any effects they may have on fibril formation. This chapter details the methods used for expression and purification of recombinant constructs of mouse, hamster and elk prions, as well as their biophysical

characterisation in order to confirm their identity, purity and structure for subsequent use in research detailed in experimental chapters of the thesis.

44

Figure 2.1 Ribbon representation (A) and space-filling model (B) of the structure of the C-terminal globular domain of mouse prion protein (residues 90–230) based on the NMR structure (Gossert et al. 2005). Main structural features are highlighted in colour with key residues shown using stick representation. Three main α-helices are shown in orange, with short antiparallel β-sheet S highlighted in blue. Native tryptophan residues at positions 98 and 144 are highlighted in green and phenylalanine residue at position 197 is highlighted in red.

The NMR structure was drawn from PDB file 1XYX using UCSF Chimera package from the Resource for Biocomputing, Visualization and Informatics at the University of Califormia, San Francisco (supported by NIH P41 RR-01081).

A

45

Figure 2.2 Ribbon representation (A) and space-filling model (B) of the structure of the C-terminal globular domain of Syrian hamster prion protein (residues 90–231) based on the NMR structure (James et al. 1997). Main structural features are

highlighted in colour with key residues shown using stick representation. Three main α- helices are shown in orange, with short antiparallel β-sheet S highlighted in blue. Native tryptophan residues at positions 99 and 145 are highlighted in green and phenylalanine residue at position 198 is highlighted in red.

The NMR structure was drawn from PDB file 1B10 using UCSF Chimera package from the Resource for Biocomputing, Visualization and Informatics at the University of Califormia, San Francisco (supported by NIH P41 RR-01081).

A

46