A method for increasing the efficiency of NMR is proposed whereby a set of 3D NOESY experiments which employ heteronuclear labelling in both indirect dimensions are used to aid assignment of standard 3D ¹⁵N- and ¹³C-edited NOESY spectra. Using these experiments in concert allows more precise identification of the ¹H nuclei involved in NOE interactions and, in doing so, ambiguity in the assignment of NOESY peaks is significantly reduced or eliminated. The complementary experiments investigated are a 3D ¹³C, ¹⁵N-HSQC-NOESY-HSQC and 3D ¹⁵N, ¹⁵N-HSQC-NOESY-HSQC for use with 3D ¹⁵N-edited NOESY and 3D ¹³C, ¹³C-HSQC-NOESY-HSQC for use with 3D ¹³C-edited NOESY. A methyl selective experiment, 3D ¹³C, ¹³CH₃-HSQC-NOESY-HSQC for use with 3D ¹³CH₃-edited NOESY is also included. Pulse programs for these sequences have been written and implemented. Semi-automated assignment protocols, developed within the CCPNMR Analysis software package, are developed and used to allow effective manipulation and elucidation of the multiple spectra, streamlining the process. The procedure is tested on the 18 kDa protein, β-Lactoglobulin B and the 15 kDa protein C4BP~1,2. The extent to which assignment ambiguity is reduced relative to a standard assignment method and the effect upon the time requirements for structure determination is investigated. It is shown that, for the sizes of proteins investigated, the time needed to obtain solution structures is reduced from months to weeks. During the course of this work, a near-complete resonance assignment of the protein β-Lactoglobulin B is achieved using standard triple resonance experiments for backbone and sidechain assignment and the low pH solution structure of this protein is solved.

NMR spectroscopy can be used to investigate the structure and dynamics of proteins, both of which can provide important insights into their functions. The aims of this project were to use residual dipolar coupling (RDC) restraints to refine the existing NMR structures of several proteins and to use relaxation data to investigate their dynamics. Three proteins were studied: two module-pair fragments from human complement regulatory proteins (DAF ~ 2,3 and C4BP~1,2) and a fatty acid binding protein from the nematode parasite <i>Ascaris suum</i> (ABA-1A), each of which consists of two structural elements, with a poorly defined relative orientation. Implementing experiments required for the measurement of coupling constraints led to the development of new methods for the determination of the one-bond ¹<i>D</i>_CαHαRDC. Extensive sets of RDCs were measured for C4BP~1,2 and ABA-1A using ¹⁵N and ¹³C labelled samples and the structure refinement protocol incorporating RDCs as restraints was optimised. Significant improvements in the quality of the refined structures were achieved. The backbone dynamics of DAF~2,3 and C4BP~1,2 were probed using relaxation data measured at two fields and the time scale and extent of back-bone motions were quantified. Finally, some evidence of small amplitude intermodular motions in the module-pair proteins DAF~2,3 and C4BP~1,2 was observed. The functional significance of these findings is discussed.

Flavocytochrome <I>b</I><SUB>2</SUB> from <I>Saccharomyces cerevisiae</I> is a L-lactate:cytochrome <I>c</I> oxidoreductase found in the mitochondrial intermembrane space. The crystal structure has been solved to 2.4Å resolution showing the enzyme to be a homotetramer (M<SUB>r</SUB> 230 000). Each subunit (511 amino acids) is composed of two domains: A cytochrome domain containing protohaem IX (residues 1-100); and a flavodehydrogenase domain, FDH, which contains FMN (residues 101-511). The two domains are tethered by a "hinge" region (residues 92-103). In the crystal structure residues 295-311 are highly disordered and so not visible. This region is a proteolytically sensitive loop for which a function has not yet been determined. Flavocytochrome <I>b</I><SUB>2</SUB> has been fully characterised and shown to be a "biological transformer" accepting electrons from a strictly 2-electron donor, L-lactate, and passing them individually to a 1-electron acceptor, cytochrome <I>c</I>. FDH has been independently expressed in E.coli allowing accurate characterisation of its properties without interference from the intense absorbance of the cytochrome domain. This has shown that although FDH remains an efficient L-lactate dehydrogenase, it has negligible cytochrome <I>c</I> reductase activity. This is intriguing as the thermodynamic driving force for electron transfer from FDH to cytochrome <I>c</I>, as determined from reduction potentials, is greater than 1/3V. We believe a lack of inter-protein recognition between the two proteins prevents them forming an effective electron transfer complex. The aim of this work was to alter the electron acceptor specificity of FDH to favour interactions with positively charged molecules.

Bacterial resistance to standard antibiotic therapies is a critical health protein worldwide. Antimicrobial-resistant infections that are essentially untreatable have begun to occur as epidemics around the globe. In this thesis, I have used the techniques of coordination chemistry, molecular biology, and protein x-ray crystallography to study metal binding of ferric ion binding protein (Fbp). Fbp is a highly-conserved 34 kDa periplasmic iron transporter, a candidate as target for novel metalloantibiotic design. I have discovered that the characteristic dityrosyl motif of Fbp from the human pathogen <i>Neisseria gonorrhoeae</i> can assemble/bind metal clusters in the open metal-binding cleft of the protein, although closure of the hinged metal-binding cleft and the presence of the synergistic phosphate anion are usually considered obligatory for strong metal binding by Fbp. I have also observed that the phosphate ion in the crystal structure of <i>apo</i>-Fbp can be displaced by mononuclear [Hf(NTA)₂]^2-, mononuclear [Fe(NTA)₂]^3- and binuclear [Fe₂(cit)₂(H₂O)₂]^2-, yielding a range of oxo Hf(IV) clusters containing from 3 to 5 metal ions, and oxo Fe(III) clusters containing 3 metal ions. Structural comparisons among the crystal structures of <i>apo</i>-Fbp (1.9 A resolution) and the Hf-bound form of Fbp (1.7 A resolution) and Fe-NTA-Fbp (1.8 A resolution) and Fe₃-FBP (1.8 A resolution) show no hinge closure upon metal binding, suggesting a novel metal acquisition mechanism. The binding of vanadate to <i>apo</i>-FBP has been studied by ⁵¹V NMR, and the data suggest that vanadate can bind similarity to phosphate and also as a vanadium cluster. Crystals of V-Fbp were obtained which diffract to 2.0 A. The work reveals the possibility that bacterial metal uptake is mediated by novel Fbp intermediates including cluster adducts.

Bovine conglutinin is a member of the collectin family of innate immune system proteins. Crystals of the native protein have been grown and X-ray diffraction data collected. The space group is tetragonal P4) with one CRD per asymmetric unit. The crystallographic structure of a recombinant bovine conglutinin carbohydrate recognition domain (CRD) has been solved to 1.2SA in native and 1.46A in ligand-bound form. The native structure has been solved by molecular replacement with the coordinates of oneCRD of human trimeric surfactant protein-D (hSP-D) as the search" model. hSP-D is the closest molecule to conglutinin phylogenetically for which the crystallographic structure is known. The fold is demonstrated to be the same as the highly conserved C-type lectin fold found in coUectins. The ligand bound structure was solved by soaking N-acetylglucosamine, the highest affmity ligand, into native crystals. Binding is shown to be by vicinal equatorial hydroxyl groups on the sugar ring of the ligand, confirming the carbohydrate recognition previously demonstrated for hSP-D and rat mannose binding proteins A and C. An additional hydrogen bond is observed between the acetyl group of the ligand and a protein lysine residue which could account for the high affinity of conglutinin for this ligand. The fmal R-factors are Rc:onv 21.7, Rrn,., 24.3 for the native, and ~v 20.3, Rne 22.3 for the ligand-bound structure. Attempts were made to grow crystals of a trimeric conglutinin fragment consisting of the a-helical coiled-coil neck region and three eROs. The crystals grown did not diffract to high enough resolution to allow data collection and structure solution. Reports that N-acetylmuramic acid, a component of bacterial cell wall peptidoglycan, was a high affinity ligand for hSP-D were followed up. Cocrystailisation trials failed to grow diffraction quality crystals.

Fourier transform infrared spectroscopy (FTIR) has been used to study a range of different proteins. These include:- i) Human serum transferrin, human lactoferrin and rabbit serum transferrin. A study has been made of the structural changes that accompany iron binding and release from these related proteins. Structural variations within this group of proteins have been shown. Thermal denaturation studies, using differential scanning calorimetric measurements, have been related to FTIR spectral changes and indicate that the crystal structure of iron-free human lactoferrin may not reflect the structure of this protein in solution. ii) Human placental transferrin receptor. This protein has been examined at extracellular and endosomal pH. Both the intact protein in detergent and its water soluble major extracellular fragment have been investigated. iii) The bacterial adhesive protein streptococcal antigen I/II. The solution structure and thermal stability of the native protein, the recombinant full-length protein and four recombinant fragments of this protein have been examined using both Fourier transfonn infrared spectroscopy and circular dichroism spectroscopy. iv) Albumin, IgG, Ribonuclease, Fibrinogen. A study of the thermal stability of these and other proteins has been undertaken to compare their denaturation properties in H20 and 2H20 solutions. v) Lysozyme, Ribonuclease. These and several other proteins have been examined using Fourier transfonn infrared microscopy in order to compare the infrared spectra of these proteins, both in solution and also in the fonn of single crystals. Many of the proteins show similar spectra, whether recorded in solution or from a single crystal. In some cases, as with single crystals of Endothia parasitica pepsin, Mucor pucillus pepsin and serum amyloid P component, the spectra differ from those recorded in solution. These differences may indicate that a rearrangement of turns structures occurs upon crystallisation. The major novel findings of this work are: i) There are small but significant structural differences between human serum transferrin, rabbit serum transferrin and human lactoferrin. The crystal structure of human lactoferrin may not represent the structure of this protein in solution. The apparent conflict between CD, FTIR and X-ray crystallographic estimates of secondary structural content of the transferrins can be explained by the fact that CD estimates are based on dihedral angles and not hydrogen bonding patterns. ii) Intact transferrin receptor precipitates out of solution at endosomal pH while the extracellular fragment remains soluble but undergoes a confonnational change. This results in minor change in the secondary structural content of the protein and a reduction of thennal stabi lity by approximately 15°C. iii) The recombinant polypeptides produced to study the structure of streptococcal antigen IIII have been shown to fold into defined secondary structures. Furthermore the structures of these fragments have been used to predict a possible structure for the native protein. iv) Quantitative analysis of thermally induced changes in the FTIR spectra of proteins in H20 solution may not offer any significant advantage to analyses performed in 2H20 due to problems associated with the different molar absorption coefficients of the separate secondary structures in H20 solution. v) Infrared spectroscopy/microscopy can be successfully be applied to the study of protein structure in both the solution and crystalline form.

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