There is an important need to immobilise enzymes for use in industry, to do this I have the promising idea that by conjugating the enzyme to a hydrogel network, thus fabricating a stable biocatalytic network would be a potential method for immobilising enzymes for the manufacture of fine chemicals, this has not been done before for octapeptide systems. Hydrogels have been previously shown as a viable way of immobilising and stabilising enzymes. In this thesis the octapeptide VKVKVEVK (V is valine, K is lysine and E is glutamic acid) is used to immobilise enzymes tagged with VKVKVEVK. This peptide sequence is chosen as it forms stable hydrogels at enzyme appropriate conditions (pH 7). The enzymes chosen are; PETNR as it is well understood and is therefore a good starting point, CDH and CHMO were also chosen as they could combine with PETNR to form a cascade reaction. PETNR was both chemically conjugated to VKVKVEVK (SpepPETNR) and also genetically modified to express the peptide tag (CpepPETNR), whilst CDH and CHMO were genetically modified to express the tag (NpepCDH and CpepCHMO respectively). For S/CpepPETNR retention within the hydrogels was superior to retention for untagged PETNR. NpepCDH was found to not precipitate within the hydrogel whilst untagged was found to do so. CpepCHMO functionalised hydrogels were found to be heterogeneous. Characterisation of CpepPETNR functionalised hydrogels was undertaken using micro differential scanning calorimetry (µDSC), rheology, small angle neutron scattering (SANS) and atomic force microscopy (AFM). From the µDSC evidence of 'protective immobilisation' was observed by the increase in denaturation energy (+253 kJ mol-1) in the hydrogel in comparison to in solution (+18 kJ mol-1). The ability of S/CpepPETNR functionalised hydrogels to perform the ketoisophorone to levodione biotransformation reaction was explored with yields of 86%. S/CpepPETNR within VKVKVEVK hydrogels was found to retain ~90% conversion for at least 9 months at room temperature. Incubation overnight at 90°C resulted in a yield of 84% of levodione. These two results added more evidence for 'protective immobilisation'. Hydrogels functionalised with NpepCDH or CpepCHMO were characterised using rheology and atomic force microscopy. The biotransformation ability of NpepCDH was elucidated; the overall yield of carvone was a maximum of 54% from the hydrogel phase. NpepCDH was used alongside CpepPETNR for the cascade reaction producing dihydrocarvone in low yields; however, an improvement from 2% to 13% in yield is presented. The yield of lactone products from CpepCHMO functionalised hydrogel was low at 15%. The CpepPETNR/ CpepPETNR cascade reaction proceeded with a yield of 36%. The initial activities of CpepPETNR, NpepCDH and CpepCHMO were assayed in both solution and in gel phase using a modified method. The activities were assessed with varying conditions; temperature, pH, quantity of ethanol and incubation at high and low temperatures. Generally, it was found that immobilisation within the hydrogel phase resulted in 'protective immobilisation' against non-optimal conditions. This work will be of benefit to those who are interesting immobilising enzymes within hydrogels in the future.

Den kopparbindande egenskapen hos prionproteiner är sannolikt kopplad till proteinets funtion. Det mänskliga prionproteinet innehåller ett kopparbindande oktapeptidområde, där PHGGGWGQ-sekvensen upprepas fyra gånger i följd. Syftet med detta arbete är att undersöka strukturen och dynamiken i oktapeptidområdet genom att använda teoretiska metoder. Med kvantkemisk strukturoptimering genomfördes en detaljerad jämförelse av växelverkan mellan flera katjoner och det kopparbindande området. Enligt dessa beräkningar är rodium(III) en möjlig ersättare för koppar(II) i NMR-specktroskopiska koordinationsstudier. Alternativa solvatiseringsmodeller i molekyldynamiksimuleringar har också jämförts. Periodiska randvillkor är mest lämpade för användning i de system som undersöks i detta arbete. Molekyldynamiksimuleringar användes för att jämföra oktapeptidområdets struktur och dynamik med och utan kopparjoner. Växelverkan mellan aminosyrornas ringar påverkar starkt strukturen i detta område i frånvaro av kopparjoner. Fyra olika icke-bindande och bindande modeller har studerats, vilka skiljer i sin beskrivning av växelverkan mellan koppar och proteinet. Teoretiska EXAFS spektra beräknades från dem simulerade strukturerna. Spektra som genererats för den bindande modellen är nästan identiska med experimentiella resultat, vilket stöder användandet av den bindande modellen. Detta arbete visar att kopparjoner interagerar med histidin imidazolringens Nδ, deprotonerade amidkväven hos de därpå följande glycinerna samt karbonylsyret hos den andra glycinen. Simuleringarna kunde visa att kopparjonen inte stabilt binder några axiella vattenmolekyler i lösning, till skillnad från en kristallstruktur av koordinationsstrukturen. Indolringen hos tryptofan interagerar direkt med kopparjonen genom stabiliserande katjon-π växelverkan utan direkt medverkan av någon vattenmolekyl. Växelverkan mellan indolringen och kopparjonen var väldefinierad och observerades kunna ske på båda sidor av koordinationsplanet. Molekyldynamiksimuleringarna med kopparjoner och oktapeptidområdet visade hur närvaron av kopparjoner ledde till ett mer strukturerat oktapeptidområde. / The copper-binding ability of the prion protein is thought to be closely related to its function. The human prion protein contains a copper-binding octapeptide region, where the octapeptide PHGGGWGQ is repeated four times consecutively. This work focuses on investigation of the structure and the dynamics of the octapeptide region by means of theoretical methods. Quantum chemical structural optimization allowed a detailed comparison of the interaction of several cations at the copper coordination site. These calculations identified rhodium(III) as a potent substitute for copper(II) that could be used to study the coordination site with NMR-spectroscopic methods. Solvation models that could be used in molecular dynamics simulations as an alternative to periodic boundary conditions were evaluated. Periodic boundary conditions are the best method for modeling the aqueous bulk in the kind of systems that are studied in this work. Molecular dynamics simulations were used to compare the behavior of the octapeptide region in the absence and presence of copper ions. Interaction between nonpolar rings strongly influences the structure of the region in the absence of copper ions. Four different non-bonded and bonded models for describing the interaction between copper and the protein were evaluated. Theoretical EXAFS spectra were calculated from the simulated structures. The results obtained for the bonded model are nearly identical with experimental data, which validates the use of the bonded model. This work thus shows strong evidence for copper(II) ions interacting with the octapeptide region through the histidine imidazole Nδ, the deprotonated nitrogen atoms of the following two glycine residues and the carbonyl oxygen atom of the second glycine residue. Notably, the simulations show that the axial sites of the copper ion do not stably coordinate water molecules in solution, as opposed to the crystal structure reported for the coordination site. Instead, the tryptophan indole ring interacted directly with the copper ion through stabilizing cation-π interaction without water mediation. The interaction of the indole ring with the copper ion was well-defined and was observed to occur on both sides of the coordination plane. The investigations of the interaction between copper ions and the octapeptide region with molecular dynamics simulations show how the presence of copper ions results in a more structured octapeptide region. / QC 20100816