The work described in this thesis is concerned with the development and application of real-time photo-CIDNP (Chemically Induced Dynamic Nuclear Polarisation) to the study of protein structure and folding. Chapters 1 and 2 introduce the protein folding problem, and its study by NMR, then go on to elucidate the mechanisms behind the photo-CIDNP phenomenon. Chapter 3 applies photo-CIDNP spectroscopy to the study of a small cytochrome protein. The difficulties of performing these experiments on chromophore-containing proteins are discussed. Chapter 4 begins with the development of a rapid mixing device for use in real-time NMR and CIDNP studies. Experiments used to characterise the device are presented. This chapter then goes on to describe CIDNP pulse labelling experiments, used to investigate the surface structure of some molten globule states of two a-lactalbumins. This chapter concludes with an application of the rapid mixing device to the real-time refolding of hen egg white lysozyme. Chapter 5 extends the work of the previous chapter, studying the real-time refolding of bovine pancreatic ribonuclease A. Refolding studies are performed from different denaturing conditions, and the effects of sample heating during the real-time CIDNP experiment are discussed. Chapter 6 describes the use of illumination during an NMR experiment to study the conformational changes in a plant blue light receptor protein, phototropin. The structural changes are characterised with 2-dimensional NMR spectroscopy and photo-CIDNP. The kinetics of the ground state recovery are also investigated by real-time NMR spectroscopy. Chapter 7 uses calculated hyperfine coupling constants and a radical pair diffusion model from the literature to simulate the nuclear polarisation obtained for the amino acid tryptophan. Comparisons are made between theory and experiment. Chapter 8 describes the structural characterisation of a homologous series of de novo peptides, designed for subsequent use in EPR experiments when derivatised with a suitable spin label.

The calcium binding epidermal growth factor-like (cbEGF) 12-13 domain pair from human fibrillin-1 was the focus of studies for this dissertation. Various nuclear magnetic resonance (NMR) spectroscopy techniques were employed to analyse the calcium binding, structural and dynamic properties of this pair, and to assess the effects of a disease-causing mutation. Fibrillin-1 is a mosaic protein composed mainly of 43 cbEGF domains arranged as multiple, tandem repeats, and mutations within fibrillin-1 have been linked to Marfan syndrome (MFS). 66% of MFS-causing mutations identified thus far are localised to cbEGF domains, emphasising that the native properties of these domains are critical to the functional integrity of this protein. The cbEGF 12-13 pair is found within the longest run of cbEGFs in fibrillin-1, and many mutations that cluster in this region are associated with the severe, neonatal form of MFS. It is thought that this region may be important for fibrillin-1 assembly into 10- 12nm connective tissue microfibrils. Calcium binding studies of cbEGF 12-13 demonstrated that cbEGF 13 contains the highest affinity site thus far investigated from human fibrillin-1. Comparison with previous results showed that fibrillin-1 cbEGF calcium binding affinity can be significantly modulated by the type of domain which is linked to its N-terminus, and also highlighted the high affinity of the "neonatal" region. The NMR solution structure of cbEGF 12-13 is a near-linear, rod-like arrangement of two cbEGF domains, with both exhibiting secondary structure characteristic of this domain type. The rod-like arrangement is stabilised by calcium binding by cbEGF 13 and by hydrophobic interdomain packing interactions. This observation supports the hypothesis that all Class I EGF/cbEGF-cbEGF pairs, characterised by a single linker residue, possess this rod-like structure. The structure also exhibits additional packing interactions to those previously observed for cbEGF32- 33 from fibrillin-1, which may explain the higher calcium binding affinity of cbEGF13. A model of cbEGF 11-15, created based on structural data for cbEGF 12-13 and a model of cbEGF32-36, has highlighted a potential protein binding interface, which encompasses all known neonatal MFS mutations, as well as a flexible, unstructured loop region of cbEGF 12. Backbone dynamics data confirmed the extended structure of cbEGF 12-13. These data, combined with previous data for cbEGF32-33, highlighted a potential dynamics signature for Class I cbEGF domain pairs. Comparison of data for these pairs also suggested that, in addition to the role of calcium in stabilising rigidity on the picoto millisecond time-scale, calcium affinity may play a key role in determining the anisotropy of cbEGF pairs. Possible dynamic explanations for the variation in calcium binding affinity of cbEGF domains from human fibrillin-1 were also noted. The Gl 127S mutation located in cbEGF 13 of fibrillin-1 causes a mild variant of MFS. NMR studies of the G1127S cbEGF12-13 mutant pair showed that cbEGF12 may chaperone folding of mutant cbEGF 13, an effect most likely mediated through interdomain packing interactions. These studies have also shown that the effects of this mutation are localised to cbEGF13, suggesting that a "partial" MFS phenotype is the result of altered structural, dynamic and/or calcium binding properties of this domain.

This thesis describes the use of nuclear magnetic resonance techniques to determine the structures of two proteins in solution, hen egg-white lysozyme and human interleukin-4. Using 2D ¹H methods an extensive set of structural data has been collected for hen lysozyme (1158 NOE distance restraints, 68 o and 24 _?1 dihedral angle restraints) and these data have been used in distance geometry-dynamical simulated annealing calculations to determine an ensemble of NMR structures for the protein. The overall Ca RMSD from the average for a set of 16 calculated structures is 1.8 ± 0.2 A but, excluding 14 residues in exposed disordered regions, this value reduces to 1.3 ± 0.2 Å. Regions of secondary structure, and particularly the four a helices, are well defined (Ca RMSD 0.8 ± 0.3 Å for helices). Detailed comparisons of the NMR structures with crystal structures of the protein have shown the close similarity of the main chain fold and the conformation of interior side chains in solution and in crystals. ³J_aß coupling constant measurement have indicated, however, that the conformational mobility of the side chains of many surface residues is significantly more pronounced than an individual crystal structure would suggest. For human interleukin-4, a strategy involving ¹⁵N and ¹³C labelled recombinant protein together with heteronuclear 3D NMR techniques has been employed to determine the structure of the protein. The work has provided the first structure for this protein, a left-handed four helix bundle with an up-up-down-down connectivity. For an ensemble of 10 final calculated NMR structures there is a Ca RMSD from the average of 1.6 ± 0.4 Å, the definition of the helical core of the protein being particularly good (0.8 ± 0.2 Å). There is, however, some disorder in the long overhand loops of the structure; this reflects the unusually high conformational mobility of these regions. Comparison of the interleukin-4 structure with proteins with related folds, particularly members of the haemopoietic cytokine superfamily, suggests that the fold found here for interleukin-4 may be the adopted structure throughout this cytokine superfamily. In a postscript to this thesis the NMR structure of human interleukin-4 is shown to have a very similar conformation to a crystal structure of the protein which has been solved very recently.

Most cellular magnesium is bound, yet it is the concentration of free magnesium, [Mg²⁺]_free, in red blood cells that is vital in the regulation of enzyme activity and ion transport. It is unknown how changes in total blood magnesium affect the [Mg²⁺]_free within red blood cells or in tissue, because the presence of other cations, especially H⁺ and potassium, K⁺ , affects the degree to which Mg²⁺ is bound. Consequently, this Thesis presents a new ³¹P NMR spectroscopic method to measure [Mg²⁺]_free in blood, which analyses the changes in the phosphorus chemical shifts of ATP and 2,3-DPG using theoretical equations expressing the observed chemical shift as a function of pH, K⁺ and [Mg²⁺]_free, over the pH range of 5.75 to 8.5 and [Mg²⁺]_free range 0 to 5 mM. The equations were adjusted for the binding of haemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K⁺ and Hb. These equations enabled, for the first time, the simultaneous analyses of the chemical shifts of 3P-DPG and β-ATP to measure both intracellular 04- pH and [Mg²⁺]_free in normal and sickle blood. To simulate in vivo 100% oxygenated blood, samples were prepared for analysis by equilibration with a mixture of O₂ and CO₂, adjusted to give a pCO₂ of 40 mmHg and pO₂ > 150 mmHg. Under these conditions, normal whole blood had an intracellular pH of 7.20 ± 0.02 and a [Mg²⁺]_free of 0.41 ± 0.03 mM (n = 33). Further work determined blood pH and [Mg²⁺]_free for several clinical conditions including sickle cell anaemia, pre-eclampsia, hypoxia, patients with sub-arachnoid haemorrhage and chronic fatigue syndrome. This Thesis has demonstrated the potential of this new technique to evaluate the importance of [Mg²⁺]_free in the regulation of metabolite concentration and metabolic function, and to elucidate some of the properties of magnesium transport across the erythrocyte cell membrane.

Some physicochemical properties of several series of transition metal chalcocarbonyls such as ((eta)('6)-C(,6)H(,6))Cr(CO)(,2)(CX) and Cr(CO)(,5)(CX) (X = O, S, Se) have been investigated. In particular, the infrared, ('13)C and ('17)O nuclear magnetic resonance and ultraviolet spectra have been examined. The results show that the order of (sigma)-donor and (pi)-acceptor capabilities of the ligands is CO CS. The M((pi)) (--->) CX((pi)*) ultraviolet charge-transfer bands are shown to correlate with the respective (mu)'(,MCX) and ('13)C NMR data. Also, the ('13)C NMR chemical shifts and GQVFF force constants are found to be highly correlated. The ('17)O NMR spectra of the metal chalcocarbonyl complexes display chemical shifts in the opposite direction to the corresponding ('13)C ones. No correlation is found between the ('17)O shieldings and the other spectroscopic data. Reaction of Cr(CO)(,5)(CX) (X = S, Se) with halide ions (Y('-)) afforded mixtures of {Cr(CO)(,5)Y}('-) and trans-{Cr(CO)(,4)(CX)Y}('-) while Cr(CO)(,5)(CS) reacted with cyclohexylamine to give Cr(CO)(,5)(CNC(,6)H(,11)). The similarities and differences in the physicochemical behaviour of the metal chalcocarbonyls compared to related systems are discussed in the light of the different bonding patterns.

The three-dimensional structure of a protein molecule is the key to understanding its biological and physiological properties. A major problem in bioinformatics is to efficiently determine the three-dimensional structures of query proteins. Protein NMR structure de- termination is one of the main experimental methods and is comprised of: (i) protein sample production and isotope labelling, (ii) collecting NMR spectra, and (iii) analysis of the spectra to produce the protein structure. In protein NMR, the three-dimensional struc- ture is determined by exploiting a set of distance restraints between spatially proximate atoms. Currently, no practical automated protein NMR method exists that is without human intervention. We first propose a complete automated protein NMR pipeline, which can efficiently be used to determine the structures of moderate sized proteins. Second, we propose a novel and efficient semidefinite programming-based (SDP) protein structure determination method. The proposed automated protein NMR pipeline consists of three modules: (i) an automated peak picking method, called PICKY, (ii) a backbone chemical shift assign- ment method, called IPASS, and (iii) a protein structure determination method, called FALCON-NMR. When tested on four real protein data sets, this pipeline can produce structures with reasonable accuracies, starting from NMR spectra. This general method can be applied to other macromolecule structure determination methods. For example, a promising application is RNA NMR-assisted secondary structure determination. In the second part of this thesis, due to the shortcomings of FALCON-NMR, we propose a novel SDP-based protein structure determination method from NMR data, called SPROS. Most of the existing prominent protein NMR structure determination methods are based on molecular dynamics coupled with a simulated annealing schedule. In these methods, an objective function representing the error between observed and given distance restraints is minimized; these objective functions are highly non-convex and difficult to optimize. Euclidean distance geometry methods based on SDP provide a natural formulation for realizing a three-dimensional structure from a set of given distance constraints. However, the complexity of the SDP solvers increases cubically with the input matrix size, i.e., the number of atoms in the protein, and the number of constraints. In fact, the complexity of SDP solvers is a major obstacle in their applicability to the protein NMR problem. To overcome these limitations, the SPROS method models the protein molecule as a set of intersecting two- and three-dimensional cliques. We adapt and extend a technique called semidefinite facial reduction for the SDP matrix size reduction, which makes the SDP problem size approximately one quarter of the original problem. The reduced problem is solved nearly one hundred times faster and is more robust against numerical problems. Reasonably accurate results were obtained when SPROS was applied to a set of 20 real protein data sets.

NMR spectroscopy is not only for protein structure determination, but also for drug screening and studies of dynamics and interactions. In both cases, one of the main bottleneck steps is backbone assignment. When a homologous structure is available, it can accelerate assignment. Such structure-based methods are the focus of this thesis. This thesis aims for fast and robust methods for NMR assignment problems; in particular, structure-based backbone assignment and chemical shift mapping. For speed, we identified situations where the number of 15N-labeled experiments for structure-based assignment can be reduced; in particular, when a homologous assignment or chemical shift mapping information is available. For robustness, we modeled and directly addressed the errors. Binary integer linear programming, a well-studied method in operations research, was used to model the problems and provide practically efficient solutions with optimality guarantees. Our approach improved on the most robust method for structure-based backbone assignment on 15N-labeled data by improving the accuracy by 10% on average on 9 proteins, and then by handling typing errors, which had previously been ignored. We show that such errors can have a large impact on the accuracy; decreasing the accuracy from 95% or greater to between 40% and 75%. On automatically picked peaks, which is much noisier than manually picked peaks, we achieved an accuracy of 97% on ubiquitin. In chemical shift mapping, the peak tracking is often done manually because the problem is inherently visual. We developed a computer vision approach for tracking the peak movements with average accuracy of over 95% on three proteins with less than 1.5 residues predicted per peak. One of the proteins tested is larger than any tested by existing automated methods, and it has more titration peak lists. We then combined peak tracking with backbone assignment to take into account contact information, which resulted in an average accuracy of 94% on one-to-one assignments for these three proteins. Finally, we applied peak tracking and backbone assignment to protein-ligand docking to illustrate the potential for fast 3D complex determination.

Includes bibliographies / x, 149 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986

Mitochondria evolved through endosymbiosis of an ancient prokaryote, and subsequently lost most genes to the host genome. In order for mitochondrial proteins to be correctly localized from the host cytosol to the mitochondrial compartments, a complex protein targeting and import machinery has evolved. Key receptor components in the protein translocase complex of the outer mitochondrial membrane, Tom20 and Tom22, recognize proteins to be imported and assist their insertion across the outer membrane. The solution structure of the Tom20 receptor domain from Arabidopsis thaliana was determined by nuclear magnetic resonance spectroscopy, and revealed that this protein has significant structural differences to its functional analogue found in animals and fungi.

Homocysteine, an independent risk factor for cardiovascular disease, is methylated in the liver via the zinc metalloenzyme betaine-homocysteine methyltransferase (BHMT). Established assays for BHMT include a radiochemical assay, a colorometric assay, an HPLC assay and an in vivo microbiological assay. These techniques are either unsuitable for substrate specificity studies, or are unable to give kinetic measurements. BHMT was purified from liver and measured directly and kinetically by a novel ¹H-NMR spectroscopic assay. The disappearance of substrates and the formation of products are monitored simultaneously. Using 2 mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH = 7.5) and measuring the production of N,N-dimethylglycine the CV is 6.3% (n = 6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed and had CV = 5.3%, n = 6, with a detection limit of 2 nkatal. The NMR spectroscopic assay was used to determine the substrate specificity with a library of alternative substrates. Analysis of betaine analogues with different chain length, α-substitution, substitution of the nitrogen and carboxyl moieties demonstrated that BHMT is inactive if there is any steric crowding of the nitrogen or α-carbon positions. BHMT is capable of using group VI heteroatom betaines as methyl donors, with much faster rates than glycine betaine. For glycine betaine the Km was 0.19 ± 0.03 mM with a Vmax of 17 ± 0.7 nMol min-1 mg-1. The same assay was used to detect and partially characterise a BHMT activity from hagfish liver that is similar to that of the mammalian enzyme. NMR spectroscopy was adapted for measurements of glycine betaine in urine, along with other medically significant methylamines. These were shown to be valid for clinical use and in animal studies. A novel metabolite of the sulfonium analogue of glycine betaine (methylsulfinylmethanoate) was identified in rats.