<p>Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases.</p><p>The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A.</p><p>Finally, the structure of YegS, an <i>Escherichia coli</i> protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.</p>

Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases. The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A. Finally, the structure of YegS, an Escherichia coli protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.

Das E. coli Expressionssystem ist das am häufigsten angewandte hinsichtlich der rekombinante Proteinexpression für strukturelle und funktionelle Analysen aufgrund der hohen erzielten Ausbeuten und der einfachen Handhabbarkeit. Allerdings ist insbesondere die Expression eukaryotischer Proteine in E. coli problematisch, z.B. wenn das Protein nicht korrekt gefaltet ist und in unlöslichen Inclusion Bodies anfällt. In manchen Fällen ist die Analyse von Deletionskonstrukten oder einzelnen Proteindomänen der Untersuchung des Vollängeproteins vorzuziehen. Dies umfasst die Herstellung eines Satzes von Expressionskonstrukten, welche charakterisiert werden müssen. In dieser Arbeit werden Methoden optimiert und evaluiert für die in vitro-Faltung von Inclusion Body-Proteinen sowie die Entwicklung einer Hochdurchsatz-Charakterisierung von Expressionskonstrukten. Die Überführung von Inclusion Body-Proteinen in den nativen Zustand beinhaltet zwei Schritte: (a) Auflösen mit einen chaotropen Reagenz oder starkem ionischen Detergenz und (b) Faltung des Proteins durch Beseitigung des Chaotrops begleitet von dem Transfer in einen geeigneten Puffer. Die Ausbeute an nativ gefaltetem Protein ist oft stark eingeschränkt aufgrund von Aggregation und Fehlfaltung; sie kann allerdings durch die Zugabe bestimmter Additive zum Faltungspuffer erhöht werden. Solche Additive müssen empirisch identifiziert werden. In dieser Arbeit wurde eine Testprozedur für Faltungsbedingungen entwickelt. Zur Reduzierung der möglichen Kombinationen der getesteten Additive wurden sowohl empirische Beobachtungen aus der Literatur als auch bekannte Eigenschaften der Additive berücksichtigt. Zur Verminderung der eingesetzten Proteinmenge und des Arbeitsaufwandes wurde der Test automatisiert und miniaturisiert mittels eines Pipettierroboters. 20 Bedingungen zum schnellen Verdünnen von denaturierten Proteinen werden hierbei getestet und zwei Bedingungen zur Faltung von Proteinen mit dem Detergenz/Cyclodextrin Protein-Faltungssystem von Rozema et al. (1996). 100 &#181;g Protein werden pro Bedingung eingesetzt. Zusätzlich werden acht Bedingungen für die Faltung von His-Tag-Fusionsproteinen (ca. 200 &#181;g), welche an eine Metallchelat-Matrix immobilisiert sind, getestet. Die Testprozedur wurde erfolgreich angewendet zur Faltung eines humanen Proteins, der p22 Untereinheit von Dynactin, welche in E. coli in Inclusion Bodies exprimiert wird. So wie es sich bei vielen Proteinen darstellt, war auch für p22 Dynactin kein biologischer Nachweistest vorhanden, um den Erfolg des Faltungsexperimentes zu messen. Die Löslichkeit des Proteins kann nicht als eindeutiges Kriterium dienen, da neben nativ gefaltetem Protein, lösliche fehlgefaltete Spezies und Mikroaggregate auftreten können. Diese Arbeit evaluiert Methoden zur Detektion kleiner Mengen nativen Proteins nach dem automatisierten Faltungstest. Bevor p22 Dynactin gefaltet wurde, wurden zwei Modellenzyme zur Evaluierung eingesetzt, bovine Carboanhydrase II (CAB) und Malat Dehydrogenase aus Schweineherz-Mitochondrien. Die wiedererlangte Aktivität nach der Rückfaltung wurde korreliert mit verschiedenen biophysikalischen Methoden. Bindungsstudien mit 8-Anilino-1-Naphtalenesulfonsäure ergaben keine brauchbaren Informationen bei der Rückfaltung von CAB aufgrund der zu geringen Sensitivität und da fehlgefaltete Proteine nicht eindeutig von nativem Protein unterschieden werden konnten. Tryptophan Fluoreszenzspektren der rückgefalteten CAB wurden zur Einschätzung des Erfolges der Rückfaltung angewandt. Die Verschiebung des Intensitätsmaximum zu einer niedrigeren Wellenlänge im Vergleich zum denaturiert entfalteten Protein sowie die Fluoreszenzintensität korrelierten mit der wiedererlangten enzymatischen Aktivität. Für beide Modellenzyme war analytische hydrophobe Interaktionschromatographie (HIC) brauchbar zur Identifizierung rückgefalteter Proben mit aktivem Enzym. Kompakt gefaltetes, aktives Enzym eluierte in einem distinkten Peak im abnehmenden Ammoniumsulfat-Gradienten. Das Detektionslimit für analytische HIC lag bei 5 &#181;g. Im Falle von CAB konnte gezeigt werden, dass Tryptophan-Fluoreszenz-Spektroskopie und analytische HIC in Kombination geeignet sind um Falsch-Positive oder Falsch-Negative, welche mit einem der Monitore erhalten wurden, auszuschließen. Diese beiden Methoden waren ebenfalls geeignet zur Identifizierung der Faltungsbedingungen von p22 Dynactin. Tryptophan-Fluoreszenz-Spektroskopie kann jedoch zu Falsch-Positiven führen, da in machen Fällen Spektren von löslichen Mikroaggregaten kaum unterscheidbar sind von Spektren des nativ gefalteten Proteins. Dies zusammenfassend wurde eine schnelle und zuverlässige Testprozedur entwickelt, um Inclusion Body-Proteine einer strukturellen und funktionellen Analyse zugänglich zu machen. In einem separaten Projekt wurden 88 verschiedene E. coli-Expressionskonstrukte für 17 humane Proteindomänen, welche durch Sequenzanalyse identifiziert wurden, mit einer Hochdurchsatzreinigung und &ndash;faltungsanalytik untersucht, um für die Strukturanalyse geeignete Kandidaten zu erhalten. Nach Expression in einem Milliliter im 96er Mikrotiterplattenformat und automatisierter Proteinreinigung wurden löslich exprimierte Proteindomänen direkt analysiert mittels 1D &#185;H-NMR Spektroskopie. Hierbei zeigte sich, dass insbesondere isolierte Methylgruppen-Signale unter 0.5 ppm sensitive und zuverlässige Sonden sind für gefaltetes Protein. Zusätzlich zeigte sich, dass &ndash; ähnlich zur Evaluierung des Faltungstests &ndash; analytische HIC effizient eingesetzt werden kann zur Identifizierung von Konstrukten, welche kompakt gefaltetes Protein ergeben. Sechs Konstrukte, welche zwei Domänen repräsentieren, konnten schnell als tauglich für die Strukturanalyse gefunden werden. Die Struktur einer dieser Domänen wurde kürzlich von Mitarbeitern gelöst, die andere Struktur wurde im Laufe dieses Projektes von einer anderen Gruppe veröffentlicht. / For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 &#181;g protein is used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 &#181;g) immobilised on metal chelate resins. The screen was successfully applied to fold a human protein, the p22 subunit of dynactin that is expressed in inclusion bodies in E. coli. For p22 dynactin &ndash; as is the case for many proteins &ndash; there was no biological assay available to assess the success of the folding screen. Protein solubility can not be used as a stringent criterion because beside natively folded protein, soluble misfolded species and microaggregates may occur. This work evaluates methods to detect small amounts of natively folded protein after automated folding screening. Before folding screening with p22 dynactin, two model enzymes, bovine carbonic anhydrase II (CAB) and pig heart mitochondrial malate dehydrogenase, were used for evaluation. Recovered activity after refolding was correlated to different biophysical methods. 8-anilino-1-naphtalenesulfonic acid binding-experiments gave no useful information when refolding CAB, due to low sensitivity and because misfolded protein could not be readily distinguished from native protein. Tryptophan fluorescence spectra of refolded CAB were used to assess the success of refolding. The shift of the intensity maximum to a shorter wavelength, compared to the denaturant unfolded protein, as well as the fluorescence intensity correlated to recovered enzymatic activity. For both model enzymes, analytical hydrophobic interaction chromatography (HIC) was useful to identify refolded samples that contain active enzyme. Compactly folded, active enzyme eluted in a distinct peak in a decreasing ammonium sulfate gradient. The detection limit of analytical HIC was approx. 5 &#181;g. In case of CAB, tryptophan fluorescence spectroscopy and analytical HIC showed that both methods in combination can be useful to rule out false positives or false negatives obtained with one method. These two methods were also useful to identify conditions for folding of p22 dynactin. However, tryptophan fluorescence spectroscopy can lead to false positives because in some cases spectra of soluble microaggregates are not well distinguishable from spectra of natively folded protein. In summary, a fast and reliable screening procedure was developed to make inclusion body proteins accessible to structural or functional analyses. In a separate project, 88 different E. coli expression constructs for 17 human protein domains that had been identified by sequence analysis were analysed using high-throughput purification and folding analysis in order to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, solubly expressed protein domains were directly analysed using 1D &#185;H-NMR spectroscopy. It was found that isolated methyl group signals below 0.5 ppm are particularly sensitive and reliable probes for folded protein. In addition &ndash; similar to the evaluation of a folding screen &ndash; analytical HIC proved to be an efficient tool for identifying constructs that yield compactly folded protein. Both methods, 1D &#185;H-NMR spectroscopy and analytical HIC, provided complementary results. Six constructs, representing two domains, could be quickly identified as targets that are well suitable for structural analysis. The structure of one of these domains was solved recently by co-workers, the other structure was published by another group during this project.

The fatty acid-binding protein (fabp) genes belong to the multigene family of intracellular lipid-binding proteins (iLBP). To date, 12 different FABPs have been identified in various vertebrate genomes. Owing to the fish-specific whole genome duplication (FSGD) event, many fishes have duplicated copies of the fabp genes. Here, I identified and characterized the fabp genes of spotted green pufferfish (Tetraodon nigroviridis). Initially, a BLAST search was performed and ten fabp genes were identified, out of which, three were retained in the pufferfish genome as duplicated copies. The putative pufferfish Fabp proteins shared greatest sequence identity and similarity with their teleost and tetrapod orthologs. Conserved gene synteny was evident between the pufferfish fabp genes and human, zebrafish, three-spined stickleback and medaka FABP/fabp genes, providing evidence that the duplicated copies of pufferfish fabp genes most likely arose as a result of the FSGD. The differential tissue-specific distribution of pufferfish fabp transcripts suggests divergent spatial regulation of duplicated pairs of fabp genes.

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

In 1998 the genome sequence of Mycobacterium tuberculosis H37Rv was published1. M. tuberculosis is the primary causative agent of tuberculosis, a disease with a long history in humans, which still has a great impact on human mortality today. As part of the M. tuberculosis Structural Genomics Consortium we selected nine target genes (Rv0534c (menA); Rv0548c (menB); Rv0553 (menC); Rv0555 (menD); Rv0542c (menE); Rv3853 (menG); Rv0558 (ubiE); Rv0989c (grcC2) and Rv0990c) from M. tuberculosis, including all known members of the menaquinone biosynthesis pathway, for structural studies. All nine genes were taken through the structural genomics “pipeline”, either becoming stuck at various “bottlenecks” or continuing successfully to structure solution. At the initial bioinformatics analysis step, eight of the nine targeted genes were deemed suitable for further study. PCR amplification and cloning of these genes into several different expression vectors followed. Expression of the gene products for the seven successfully cloned genes was undertaken in an E. coli expression host, followed by experiments (refolding, lysis buffer and expression temperature screens) aimed at obtaining soluble protein in sufficient quantities for crystallisation. Of the seven proteins successfully overexpressed, five remain at this stage as they could not be obtained in soluble form. The remaining two, Rv3853 (MenG), solubilised by refolding, and MenB, solubilised by 24ºC expression, were purified and both successfully produced diffracting crystals. The crystal structure of Rv3853 was determined by isomorphous replacement (SIRAS) and refined at 1.9 Å resolution (R = 19.0% and Rfree = 22.0%). The structure of several different crystal forms of MenB, were determined by molecular replacement. Refinement of two of these structures, MenB_P43212 at 2.15Å resolution (R = 20.3% and Rfree = 23.1%) and MenB_C2-NCoA at 2.3 Å resolution (R = 19.7% and Rfree = 22.5%), has been completed. The structure of Rv3853, combined with the discovery that UbiE was more likely to catalyse the final, S-adenosylmethionine-dependent, methyltransfer step of menaquinone biosynthesis, led to the conclusion that Rv3853 had been misannotated as MenG. Combined with further bioinformatics analysis the Rv3853 structure has been useful in providing new ideas as to the real function of Rv3853. In contrast, the structure of MenB confirmed its place as a member of the crotonase superfamily although the C-terminus was located in a position not observed in other crotonase superfamily structures. Several flexible regions likely to be important in MenB function have been identified by examination of the various MenB structures / Author was the recipient of a University of Auckland Doctoral Scholarship and a Foundation of Research Science & Technology Top Achiever Doctoral Scholarship

RNA-dependent RNA polymerases (RdRps) play a vital role in the life cycle of RNA viruses, being responsible for genome replication and mRNA transcription. In this thesis viral RdRps (vRdRps) of dsRNA bacteriophage phi6 (phi6 RdRp) and Severe Acute Respiratory Syndrome (SARS) coronavirus [non structural protein 12 (NSP-12)] are studied. For SARS polymerase NSP-12, a library-based screening method known as ESPRIT (Expression of Soluble Protein by Random Incremental Truncation) was employed in an attempt to isolate domains of NSP-12 that express solubly in Escherichia coli (E. coli) and are thereby suitable for structural studies. This experiment identified for the first time in a systematic fashion, conditions under which the SARS polymerase could be solubly expressed at small scale and allowed mapping of domain boundaries. Further experiments explored different approaches for increasing expression levels of tractable fragments at large scale. Bacteriophage phi6 RdRp is one of the best studied vRdRps. It initiates RNA synthesis using a de novo mechanism without the need for a primer. Although formation of the de novo initiation complex has been well studied, little is known about the mechanism for the transition from initiation to elongation (i.e. extension of an initiated dinucleotide daughter strand). In the phi6 RdRp initiation complex the C-terminal domain (CTD) blocks the exit path of the newly synthesised dsRNA which must be displaced for the addition of the third nucleotide. The crystal structure of a C-terminally truncated phi6 RdRp (P2T1) reveals the strong non-covalent interactions between the CTD and the main body of the polymerase that must be overcome for the elongation reaction to proceed. Comparing new crystal structures of complexes of both wild-type (WT) and a mutant RdRp (E634 to Q, which removes a salt-bridge between the CTD and main body of the polymerase) with various oligonucleotides (linear and hairpin), nucleoside triphosphates (NTPs) and divalent cations, alongside their biophysical and biochemical properties, provides an insight into the precise molecular details of the transition reaction. Thermal denaturation experiments reveal that Mn2+ acquired from the cell and bound at the phi6 RdRp non-catalytic ion site sufficiently weakens the polymerase structure to facilitate the displacement of the CTD. Our crystallographic and biochemical data also indicate that Mn2+ is released during this displacement and must be replaced for the elongation to proceed. Our data explain the role of the non-catalytic divalent cation in vRdRps and pinpoint the Mn2+-dependent step in viral replication. In addition, by inserting a dysfunctional Mg2+ at the non-catalytic ion site for both WT and E634Q RdRps we captured structures with two NTPs bound within the active site in the absence of Watson-Crick base pairing with template and could map movements of divalent cations during preinitiation through to initiation. Oligonucleotides present on the surface of phi6 RdRp allowed mapping of key residues involved in template entry and unwinding of dsRNA; these preinitiation stages have not been observed previously. Considering the high structural homology of phi6 RdRp with other vRdRps, particularly from (+)ssRNA hepatitis C virus (HCV), insights into the mechanistic and structural details of phi6 RdRp are thought to be relevant to the general understanding of vRdRps.

We review fold usage on completed genomes in order to explore protein structure evolution and assess the evolutionary relevance of current structural classification systems (SCOP and CATH). We assign folds on a set of 150 completed genomes using fold recognition methods (PSI-BLAST, SUPERFAMILY and Gene3D). The patterns of presence or absence of folds on genomes gives us insights into the relationships between folds and how we have arrived at the set of folds we see today. In particular, we develop a technique to estimate the relative ages of a protein fold based on genomic occurrence patterns in a phylogeny. We find that SCOP's `alpha/beta' class has relatively fewer distinct folds on large genomes, and that folds of this class tend to be older; folds of SCOP's `small protein' class follow opposite trends. Usage patterns show that folds with many copies on a genome are generally old, but that old folds do not necessarily have many copies. In addition, longer domains tend to be older and hydrophobic amino acids have high propensities for older folds whereas, polar - but non-charged - amino acids are associated with younger folds. Generally domains with stabilising features tend to be older. We also show that the reliability of fold recognition methods may be assessed using occurrence patterns. We develop a method, that detects false positives by identifying isolated occurrences in a phylogeny of species, and is able to improve genome wide fold recognition assignment sets. We use a structural fragment library to investigate evolutionary links between protein folds. We show that 'older' folds have relatively more such links than 'younger' folds. This correlation becomes stronger for longer fragment lengths suggesting that such links may reflect evolutionary relatedness.