This work is addressed to the search for functional links between GPI-protein monomeroligomer exchange and membrane dynamics and confinement. To this end, uPAR was chosen as a GPI-receptor involved in the regulation of cell adhesion, migration and proliferation. The work is focused on tracking the molecular dynamics and the homotypic interactions of uPAR, using fully fiinctional fluorescent protein-tagged chimeras expressed in HEK293 cells.

Carbohydrates are the most plentiful organic molecules on the surface of the earth and also some of the most complex. They are not just fuel sources, but also function as identifying markers for cells, on the surfaces of which they reside in the form of glycoconjugates. These identifying molecules are recognised by lectins; proteins which bind carbohydrates through non-covalent interactions. This recognition mediates a whole range of important biological processes, from fertilisation to infection with pathogens. 'Synthetic lectins' in the context of this thesis are artificially designed and synthesised organic receptor molecules. They bind sugars in aqueous environments through non-covalent interactions and as such may be considered biomimetic in the strictest sense. Synthetic lectins designed according to the 'temple' principles developed in the Davis group have so far been successful in binding sugars which have all their hydroxyl groups orientated equatorially, such as glucose. In this thesis the problem of how to design synthetic lectins for monosaccharides with less uniform distribution of functionality (for example mannose or galactose) is explored. To that end the synthesis of receptors 1 - 3 is described. Characterisation of the binding behaviour of these molecules reveals new patterns of selectivity; for example, disaccharides are bound in preference to monosaccharides. Receptor 4 shows a small but significant affinity for N-acetylneuraminic acid. This substrate is of widespread importance in physiological signalling, and few artificial receptors have previously bound this substrate in water through purely non-covalent interactions.

Calcium (Ca ²?) is a highly versatile signal that regulates a host of intracellular events across the biological spectrum. Carefully regulated changes in intracellular Ca²? concentration, over a broad temporal and spatial range, carry complex signals to a plethora of proteins who decode and transduce the information and regulate a wide variety of physiological processes.

G protein-coupled receptors (GPCRs) comprise the most "drugable" family of transmembrane proteins, GPCRs share a common structural template and a general mechanism of signal transduction, but vary greatly in sequence conservation, ligand recognition and function. The current set of class A GPCR crystal structures have facilitated the modelling of class A GPCRs. However, other classes of GPCRs have not been so easy to study. The main focus presented in this thesis is the utilisation of class A GPCR structural information to model medically important GPCRs other than class A GPCRs via molecular modelling. The lack of sequence conservation hampers modelling non-class A GPCRs using class A GPCR crystal structures. A plant GPCR, namely GCR1, has sequence homology to more than one GPCR family (class A, Band E GPCRs) and has been used to align the transmembrane region of class A and B GPCRs. Consequently, we have presented a computational protocol for the identification of putative plant GPCRs that may similarly be used to address the difficult issue of alignment between GPCR families but in this respect only GCR1 was found to be useful. GCR1 is still an orphan GPCR with no known cognate ligand. We first assessed whether GCR 1 was a valid GPCR via homology modelling and molecular dynamics. We found that GCR1 has more similarities to class A and class S GPCRs than was previously acknowledged and further support evidence that GCR1 is a GPCR. Consequently, using the class A - GCR1 - class B alignment, we have produced active and inactive homology models of the CGRP receptor, a prototypical class B GPCR. In conjunction with mutation data, these models were used to identify a number of distinct class B motifs and their class A equivalents for the first time. Finally, molecular dynamic simulations were used to further confirm the role of the class B motifs.

Lysophosphatidate (LPA) binds to a family of G-protein coupled receptors (GPCR). Three receptors belong to the endothelial differentiation gene (EDG) family, namely LPA1-3. whereas another two receptors LPA4-5 are evolutionary distant. The LPA1receptor couples to effectors via the heterotrimeric G-protein Gi to stimulate activation of the p42/p44 MAPK pathway.

Amyloid fibrils, highly ordered aggregates of mis-folded protein with a characteristic cross-beta structure, are associated with several diseases including Alzheimer's disease and the prion diseases. Amyloid fibrils are difficult to study experimentally and there is litde high resolution structural data available; therefore simulation methods have an important role to play in characterising the structures of these early species and the forces holding them together.

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No description available.

The Hsp90 based chaperone is a ubiquitous protein-folding system in the cytoplasm of eukaryotes. Several signal transduction systems and cell cycling pathways utilise an interaction with Hsp90 as an essential component. The Hsp90 chaperone is an ATP dependent chaperone, which is active as a dimer. The N- terminal domain of Hsp90 itself has very weak ATPase activity and plays an essential role in the mechanism of dimerisation. This study attempts to elucidate the nucleotide binding effects on the Hsp90 N-terminal domain by NMR. Accomplishing backbone assignments of the apo- and AMP-PNP bound forms provided a system for which individual residues could be investigated. About 200 backbone amide peaks in the HSQC were observed out of a reported 207 residues for the Hsp90 N-terminal domain. Out of these, 192 for the apo- and 182 for the AMP-PNP bound forms were assigned. Assignments were also obtained for the HN, N, C, and CO nuclei. Comparison of apo and AMP-PNP HSQC spectra showed large shift differences in areas where the nucleotide binds and in a conserved loop, which has been proposed to act as a lid to the active site. The chemical shift pattern of the AMP-PNP bound form compared to that of the ADP bound form showed a different local environment for at least 30 residues, suggesting that different nucleotide bound conformations are more than just nucleotide structural differences. Analysing the NMR results suggests that binding of AMP-PNP to the Hsp90 N-terminal domain is not sufficient to cause lid closure as previously thought. Relaxation studies highlighted regions that had different local motions in the apo- and nucleotide bound form based on individual residues within the Hsp90 N- terminal domain. The protein rotational correlation time was measured at 12.5 ns in the apo and 14 ns in the AMP-PNP bound form. No interaction between the labelled N-terminal domain and non-labelled middle domain of Hsp90 was observed. The antibiotic, novobiocin, which inhibits other members of the GHKL superfamily, was also shown not to bind to the N-terminal domain, consistent with previous studies. The study of two mutants, A107N and T101I, showed the effects of mutation in the lid region of the N-terminal domain causing chemical shifts of between 20-30 residues. Neither of these two mutants were able to bind AMP-PNP. In all nucleotide bound X-ray crystal structures solved to date, no difference between the ADP and ATP bound forms has been observed and only one conformation was found. The results presented here suggest that the structure in solution is much more variable than previously envisaged.

No description available.