Proper folding of a protein to its native state is critical for the protein to be fully functional under biological conditions. Understanding protein folding and protein folding evolution within the same structural family are key to understand which processes assist or hinder protein folding and how to prevent misfolding. Tm0979 from Thermotoga maritima, Mth1491 from Methanobacterium thermoautotrophicum and YchN from Escherichia coli belong to the homologous superfamily of YchN-like proteins (SCOP and CATH: 3.40.1260.10). The structures of these proteins have been solved as part of structural proteomics projects, which consist of solving protein structures on a genome wide scale. In solution, Tm0979 forms a homodimer whereas Mth1491 folds as a trimer and YchN is a homohexamer. The structures of the individual monomeric subunits of these three proteins have high structural similarity, despite very low sequence similarity. The biological roles of these proteins are not yet well defined, but seem to be involved in catalysis of sulphur redox reactions. This thesis focuses on characterisation of the Tm0979 homodimer and the Mth1491 homotrimer, as well as the determination of the folding mechanisms of these two proteins. The folding mechanisms of the proteins are compared to each other and to the mechanisms of other dimeric and trimeric proteins. The evolution and basis of oligomeric structure within the YchN family are analyzed. Mutations of Tm0979 and Mth1491 are designed as a basis for future work to investigate processes responsible for switches in oligomeric protein quaternary.structure.

Proper folding of a protein to its native state is critical for the protein to be fully functional under biological conditions. Understanding protein folding and protein folding evolution within the same structural family are key to understand which processes assist or hinder protein folding and how to prevent misfolding. Tm0979 from Thermotoga maritima, Mth1491 from Methanobacterium thermoautotrophicum and YchN from Escherichia coli belong to the homologous superfamily of YchN-like proteins (SCOP and CATH: 3.40.1260.10). The structures of these proteins have been solved as part of structural proteomics projects, which consist of solving protein structures on a genome wide scale. In solution, Tm0979 forms a homodimer whereas Mth1491 folds as a trimer and YchN is a homohexamer. The structures of the individual monomeric subunits of these three proteins have high structural similarity, despite very low sequence similarity. The biological roles of these proteins are not yet well defined, but seem to be involved in catalysis of sulphur redox reactions. This thesis focuses on characterisation of the Tm0979 homodimer and the Mth1491 homotrimer, as well as the determination of the folding mechanisms of these two proteins. The folding mechanisms of the proteins are compared to each other and to the mechanisms of other dimeric and trimeric proteins. The evolution and basis of oligomeric structure within the YchN family are analyzed. Mutations of Tm0979 and Mth1491 are designed as a basis for future work to investigate processes responsible for switches in oligomeric protein quaternary.structure.

This dissertation presents efforts to expand the role of mass spectrometry (MS) in structural biology. Determination of quaternary structure of a protein complex has been hindered by limited fragmentation from collision-induced dissociation (CID). As an alternative, surface-induced dissociation (SID) was implemented for a quadrupole - time-of-flight instrument in the Wysocki laboratory. This research tested the hypothesis that SID should produce fragmentation reflective of subunit organization. Furthermore, ion mobility (IM) was used to prove the direct relationship between precursor conformation and observed dissociation patterns, and the relationship between activation and product ion conformation. The structure and dynamics of a dimeric small heat shock protein (sHSP) with no solved structure was investigated. The importance of N- and C-terminal domains for dimerization was determined, and the dimers were shown to exchange subunits. From exchange kinetics it is proposed that subunit exchange is unrelated to heat shock activity. SID was used to elucidate the subunit architecture of heterogeneous protein assemblies, including one previously solved protein structure and two formerly uncharacterized proteins. The heterohexamer toyocamycin nitrile hydratase dissociated into trimers, revealing the hexamer to be a dimer of trimers. The bacterial ribonuclease toxin:antitoxin tetramer was shown to have an antitoxin dimer at its core, with flanking individual toxin subunits. The examples presented here are the first clear proof that SID results can clearly indicate the substructure of a protein assembly.IM was used to study the conformation of precursor and product ions. A greater understanding of the relationship between precursor conformation and observed dissociation patterns was developed. Different charge states of a dodecameric sHSP were found to have significantly different conformations, which were directly reflected in SID spectra. IM comparison of CID and SID product ions showed that the same charge state of a product ion from either method has the same CCS. This suggests the product ion conformation is dependent upon ion charge state, and independent of activation method and collision energy. The cause and effect relationship between precursor conformation and MS/MS patterns, and activation and product ion conformation were clearly illustrated. Together, this body of research expands the role of MS for structural biology.

Arginine kinase catalyzes the reversible phosphorylation of arginine using ATP. This phosphotransferase is found throughout invertebrate species; whereas a homologous enzyme, creatine kinase, is found in both vertebrate and invertebrate species. Arginine kinases are found as monomers of 40 kDa or 80 kDa and dimers of 80 kDa while creatine kinases are found as dimers of 80 kDa, monomers of 150 kDa, or octamers of 320 kDa. The significance or advantage of the dimeric state or various quaternary structures is still not understood for this family of enzymes. Here, arginine kinase from Isostychopus badonotus muscle was purified to homogeneity and analyzed for physical, kinetic, and immunological characteristics. The results indicate that arginine kinase from the sea cucumber, I. badonotus, is a dimer with a molecular weight of 87 kDa that displays physical and kinetic characteristics similar to other arginine kinases regardless of their weight or subunit composition. However, immunological cross-reactivity using I. badonotus polyclonal antibodies shows that dimeric arginine kinase from the sea cucumber can react with dimeric arginine and creatine kinases but not with monomeric arginine or creatine kinases. Comparable results are seen with polyclonal antibodies raised against purified monomeric arginine kinase from the American cockroach, Periplaneta americana. Monomeric arginine kinase from the cockroach reacted with monomeric arginine kinases but not with dimeric arginine or creatine kinases or monomeric creatine kinases. Arginine kinase from the sea cucumber and the cockroach is substantially inhibited by the anion nitrate which mimics the transferable phosphoryl group in the assumed rapid equilibrium, random addition reaction. Here, nitrate has been shown to inhibit both the initial velocity and percent of product formed from arginine kinase in I. badonotus and P. americana. Difference spectra for each enzyme in the presence of varying components of the transition state analog suggest that nitrate has an effect on the enzyme itself and inhibits through a mechanism beyond that of stabilization of the dead-end complex. Further characterization of the dimeric state in these enzymes on a structural level included the elucidation of the protein sequence from the American cockroach and a comparison with dimeric arginine and creatine kinases.

The oligomerisation of protein subunits is an area of much research interest, in particular the relationship to protein function. In the last decade, the potential to control the interactions involved in order to design constructs with tuneable oligomeric properties in vitro has been pursued. The subject of this thesis is the quaternary structure of members of the peroxiredoxin family, which have been seen to assume an intriguing array of organisations. Human Peroxiredoxin 3 (HsPrx3) and Mycobacterium tuberculosis alkyl hydroperoxide reductase (MtAhpE) catalyse the detoxification of reactive species, preferentially hydrogen peroxide and peroxynitrite respectively, and form an essential part of the antioxidant defence system. As well as their biomedical interest, the ability of these proteins to form organised supramolecular assemblies makes them of interest in protein nanotechnology. The work described focusses on the elucidation of the quaternary structure of both proteins, resolving previous debates about their oligomeric state. The factors influencing oligomerisation were examined through biophysical characterisation in different conditions, using solution techniques including chromatography, light and X-ray scattering, and electron microscopy. The insight gained, along with analysis of the protein-protein interfaces, was used to alter the quaternary structure through site-directed mutagenesis. This resulted in a level of control over the protein’s oligomeric state to be achieved, and novel structures with potential applications in nanotechnology to be generated. The activity of the non-native structures was also assessed, to begin to unravel the relationship between peroxiredoxin quaternary structure to enzyme activity. The formation and structure of very high molecular weight complexes of HsPrx3 were explored using electron microscopy. The first high resolution structural data for such a complex is presented, analysis of which allowed the theory of an assembly mechanism to be proposed.

This thesis examined the enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from the pathogen Mycobacterium tuberculosis. DHDPS is a validated antibiotic target for which no potent inhibitor based on substrates, intermediates or product has been found. The importance of the homotetrameric quaternary structure in E. coli DHDPS has been demonstrated by the 100-fold decrease in activity observed in a dimeric variant, DHDPS-L197Y, created by site-directed mutagenesis. This suggested a new approach for inhibitor design: targeting the dimer-dimer interface and disrupting tetramer formation. DHDPS catalyzes the first committed step in the biosynthetic pathway of meso-diaminopimelic acid, a critical component of the mycobacterial cell wall. In this study, wild-type M. tuberculosis DHDPS was thoroughly characterized and compared with the E. coli enzyme. A coupled assay was used to obtain the kinetic parameters for M. tuberculosis DHDPS: KM(S) ASA = 0.43 (±0.02) mM, KMpyruvate = 0.17 (±0.01) mM, and kcat = 138 (±2) s 1. Biophysical techniques showed M. tuberculosis DHDPS to exist as a tetramer in solution. This is consistent with the crystal structure deposited as PDB entry 1XXX. The crystal structure of M. tuberculosis DHDPS showed active-site architecture analogous to E. coli DHDPS and a dimeric variant of M. tuberculosis DHDPS was predicted to have reduced enzyme activity. A dimeric variant of M. tuberculosis DHDPS was engineered through a rationally designed mutation to analyze the effect of disrupting quaternary structure on enzyme function. A single point mutation resulted in a variant, DHDPS-A204R, with disrupted quaternary structure, as determined by analytical ultracentrifugation and gel-filtration chromatography. DHDPS-A204R was found to exist in a concentration-dependent monomer-dimer equilibrium, shifted towards dimer by the presence of pyruvate, the first substrate that binds to the enzyme. The secondary and tertiary structure of DHDPS-A204R was analogous to wild-type M. tuberculosis DHDPS as judged by circular dichroism spectroscopy and X ray crystallography, respectively. Surprisingly, this disrupted interface mutant had similar activity to the wild type enzyme, with a kcat of 119 (±6) s-1; although, the affinity for its substrates were decreased: KM(S) ASA = 1.1 (±0.1) mM, KMpyruvate = 0.33 (±0.03) mM. These results indicated that disruption of tetramer formation does not provide an alternative direction for drug design for DHDPS from M. tuberculosis. Comparison with the recently discovered dimeric DHDPS from Staphylococcus aureus shed further light on the role of quaternary structure in DHDPS. In M. tuberculosis DHDPS-A204R and the naturally dimeric enzyme, the association of monomers into the dimer involves a greater buried surface area and number of residues than found in E. coli DHDPS-L197Y. This provides a framework to discriminate which DHDPS enzymes are likely to be inactive as dimers and will direct future work targeting the dimer-dimer interface of DHDPS as an approach for drug design.