In Nature, the protein backbone is assembled from a set of 20 proteogenic amino acids. The vast diversity of the proteome is due, only in part, to the range of protein encoding genes. Additional functionalities and complexities are introduced by post-translational modification, and/or the recruitment of eo-factors, prosthetic groups and metal ions. Taking inspiration from the range of protein modifications seen in Nature, the chemical incorporation of non-natural amino acids into an enzyme's active site, for example, may enable novel mechanisms of catalysis or alter substrate specificity.

This work develops the Pl1 peptide family as new materials, utilising a strategy of mod- ifying existing peptides to tune them to specific end uses. Several peptides, based on the self-assembling, ,B-sheet forming Pl1-2 sequence, were screened as potential tissue engineering materials. This was accomplished by studying the effect of net peptide charge on aggregation and secondary structure in physiological conditions, applying both proton NMR and IR spectroscopies. Single increments of net peptide charge produce large increases in critical aggregation concentration and decreases in peptide aggregate fraction at fixed concentration. No self-assembly was detected for peptides with net charges > 3, even at high mM concentration. At least a small net charge is required for solubility. Based on these studies, a peptide with a -2 charge, Pn-4, was selected for further study. The remainder of the studies explore the use of P n-4 as a scaffold for small (peptide) and large (protein) groups. Using TEM and AFM, the self-assembly of Pn-4 has been compared to Pn-4 functionalised with bioactive RGD peptides. Two modified Pn-4 peptides (LR66 and LR46) self-assembled to some extent. Two others (LR56 and LR44) did not appear to display Pn-4-type self-assembly, though, on their inclusion in mixtures with Pn-4, the resulting structures appeared more like those of unmodified Pn-4. Finally, an amphiphilic fusion protein, KSI-(Pn-4h-His6, comprised of ketosteroid isomerase, a trimeric Pn-4 and a hexahistidine tag was investigated. KSI-(Pn-4h-His6 was sparingly soluble in H20 near neutral pH, but more soluble at low and high pH. AFM and TEM studies indicated KSI-(Pn-4h-His6 in H20 is in the form ofround particles. No Pn-4-like aggregates were observed, implying the attached protein disrupts self-assembly. Tensiometer studies at the air-water interface demonstrate that KSI-(Pn-4h-His6 is a promising protein surfactant, with surface tension reductions - at concentrations up to 1 mg ml-1 - bettering or closely matching that of the model ,B-Iactoglobulin.

In the past, the sarcomeric proteins of Caenorhabditis elegans were identified primarily by screening mutated worms for defects in locomotion. This approach excluded the identification of those proteins which are part of the sarcomeric structure, but nevertheless are not essential for the structural integrity and function of the muscle.

Beta-cell tropin (BCT) is a pro-opiomelanocortin-derived peptide which is released from the pituitary neurointennediate lobe (NIL) of rodents and other mammals. It has been shown to act as a potent insulin secretogogue, has lipogenic properties both in vivo and in vitro, and its sequence has been characterised as ACTH22•39• BCT levels have been found to be elevated in various obese/diabetic animal models, and human type II diabetes. This suggests that the peptide may have a role in the development and maintenance of the obese and diabetic states. The aim of the work described in this thesis was to characterise the mechanism of action of BCT on lipogenesis in white and brown adipose tissue, and to determine whether the hormone has a role in the pathological changes which occur during the development of dietary induced or genetic obesity. or the physiological adaptations in adipose tissue metabolism which are associated with pregnancy and lactation.

Approaches to prepare pyroglutamic acid derivatives using a chiral anthracene moiety have been carried out using a nucleophilic carboxylic acid synthon. A model study to prepare five membered ring lactams bearing a quaternary centre via a Ugi-3CR reaction using commercially available levulinic acid 162 was successful, giving the five-membered ring lactams 164a -164g in 72 - 86% yield. A Pd catalysed ring opening of an anthracene anhydride 184 cycloadduct was used to prepare a key keto-acid that unfortunately was a poor substrate for the Ugi reaction due to the geometry of keto acid to give alkene product 198. Introduction of a furan ring as a masked carboxylic acid moiety proceeded with excellent levels of diastereoselectivity, follmved by conversion into a carboxylate ester (71 - 80% yield). Flash vacuum pyrolysis (FVP) followed by reduction gave pyroglutamate esters 243 and 256 - 261 in 21 - 26% yield but poor enantioselectivity, which was found to be dependant on the electronic nature of the N-protecting group. Constructing a quaternary carbon centre using the same methodology was successful to give pyroglutamate 273 in 63% yield and 95% enantioselectivity after FVP. Other analogues gave enamides 275a-j in good to excellent yield (63 - 90%). Dimethyl dioxirane (DMDO) oxidation of enamide 275f gave hydroxy-ketone 285 in excellent yield. Subsequent double reduction with Et3SiH and NaBH4 gave two diastereoisomers in moderate yield. FVP of both diastereoisomers gave pyroglutamate ester 295 and 296 in 40% and 60% respectively.

Self-assembling peptides are currently attracting increasing interest as biological materials with a wide range of applications, including scaffolds for tissue engineering as well as drug delivery. The objective of the current work is to study the effect of amino acid size and charge distribution on the self-assembly and gelation behaviour of a series of de novo designed octapeptides.

Amino acids are essential for normal biological function. The efficient supply of amino acids is mediated by specialised membrane transport proteins. A proton electrochemical gradient provides the driving force for amino acid transport via the proton-coupled amino acid transporters PAT1 and PAT2. The objective of this study was to further our understanding of the function of PAT1 and PAT2 at the molecular and cellular level. When heterologously expressed in isolation using Xenopus laevis oocytes, radiolabelled amino acid uptakes revealed that mouse PAT1, rat PAT2 and the Drosophila CG1139 protein functioned as pH-dependent (proton-coupled), sodium-independent transporters. CG1139 closely resembled PAT1 in terms of its pH-dependence, substrate affinity (mM range) and specificity, with a notable exception being that L-serine (a relatively poor PAT1/PAT2 substrate) was an excellent CG1139 substrate. PAT1 and TauT mediate the uptake of the conditionally-essential amino acid taurine across the apical membrane of human intestinal (Caco-2) epithelial cell monolayers. Exposure of Caco-2 cell monolayers to high, dietary, concentrations of taurine reduced the transport activity of TauT but PAT1-mediated taurine uptake was maintained or increased. This may reflect a physiological adaptive response designed to optimise taurine absorption from the small intestine following ingestion of a protein-rich meal. The endogenous expression of multiple amino acid transporters with overlapping substrate specificity can constrain the study of individual transport proteins in intact tissues. Using a range of complementary techniques, 5-hydroxy-L-tryptophan and α-methyl-D,L-tryptophan were identified as novel non-transported inhibitors of PAT2. α-Methyl-D,L-tryptophan failed to inhibit PAT1 and may prove useful as an experimental tool for future studies designed to discriminate between PAT1 and PAT2 transport activity in vivo. Homology modelling of the slc36 transporters identified PAT2 residues putatively involved in: substrate binding (G67, T68, G69, E275); substrate occlusion (Y163 and F272); and stabilisation of the substrate binding site (C185 and C334). Substitution with homologous CG1139 residues conferred on the PAT2 mutants G64A and F159I the ability to transport the CG1139 substrate L-serine. Homology modelling may provide insights as to the structural defects in related SLC transporters implicated in disorders of amino acid transport.

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