The thesis describes over-expression, purification and crystallization of three proteins from Staphylococcus aureus (S. aureus). S. aureus is an important human pathogen and methicillin-resistant S. aureus (MRSA) is a serious problem in hospitals nowadays. The crystal structure of 3-Methyladenine DNA glycosylase I (TAG) was determined by single-wavelength anomalous diffraction (SAD) method. TAG is responsible for DNA repair and is an essential gene for both MRSA and methicilin-susceptible S. aureus (MSSA). The structure was also determined in complex with 3-methyladenine (3-MeA) and was solved using molecular replacement (MR) method. An assay was carried out and the molecular basis of discrimination between 3-MeA and adenosine was determined. The native crystal structure of fructose 1-phosphate kinase (PFK) from S. aureus was determined to 2.30 Å and solved using molecular replacement method. PFK is an essential enzyme involved in the central metabolism of MRSA. Despite extensive efforts no co-complex was determined, although crystals were obtained they diffracted poorly. An assay which can be used to test for inhibitors has been developed. Mevalonate Kinase (MK) is another essential enzyme in MRSA and is a key drug target in the mevalonate pathway. Native data diffracting to 2.2 Å was collected. The structure was solved using multiple isomorphorus replacement (MIR) method. A citrate molecule was bound at the MK active site, arising from the crystallization condition. The citrate molecule indicates how substrate might bind. The protein was kinetically characterized. A thermodynamic analysis using fluorescence-based method was carried out on each protein to investigate binding interactions of potential fragments and thus a drug design starting point.

A unique mechanism of protein oligomerization is domain swapping. It is a feature found in some proteins wherein a dimer or a higher oligomer is formed by the exchange of identical structural segments between protomers. Domain swapping is thought to have played a key role in the evolution of stable oligomeric proteins and in oligomerization of amyloid proteins. This thesis deals with studies to understand the significance of hinges involved in domain swapping for protein oligomerization and function. The stationary phase survival protein SurE from Salmonella typhimurium (StSurE) and Sesbania mosaic virus (SeMV) coat protein have been used as models for studies on domain swapping. This thesis has been divided into eight chapters. Chapter 1 provides a brief introduction to domain swapping, while Chapters 2 to 6 describes the studies carried out on StSurE protein, Chapter 7 deals with studies on SeMV coat protein. The final Chapter 8 provides brief descriptions of various experimental techniques employed during these investigations. Chapter 1 deals with a brief introduction to domain swapping in proteins. Examples where different domains are exchanged are cited. Then it describes physiological relevance of domain swapping in proteins and probable factors which promote swapping. Finally it also discusses the uncertainties that are inevitable in protein structure prediction and design. Chapter 2 describes the structure of Salmonella typhimurium SurE (StSurE; Pappachan et al., 2008) determined at a higher resolution. The chapter also deals with the sequence and structure based comparison of StSurE with other known SurE homolog structures. A comparative analysis of the relative conservation of N- and C-terminal halves of SurE protomer and variations observed in the quaternary structures of SurE homologs are presented. Then a brief introduction is provided on function of StSurE. The conserved active site of StSurE that might be important for its phosphatase activity is described. A plausible mechanism for the phosphatase activity as proposed by Pappachan et al. (2008) is presented. Crystal structures of StSurE bound with AMP, pNPP and pNP that was determined with the view of better understanding the mechanism of enzyme function is presented. These structures provide structural evidence for the mechanism proposed by Pappachan et al. (2008). Finally a substrate entry channel inferred from these structures is discussed. SurE from Salmonella typhimurium (StSurE) was selected for studies on domain swapping as there is at least one homologous structure (Pyrobaculum aerophilum - PaSurE) in which swapping of the C-terminal helices appears to have been avoided without leading to the loss of oligomeric structure or function. It was of interest to examine if an unswapped dimer of StSurE resembling PaSurE dimer could be constructed by mutagenesis. To achieve this objective, a crucial hydrogen bond in the hinge involved in C-terminal helix swapping was abolished by mutagenesis. These mutants were constructed with the intention of increasing the flexibility of the hinge which might bring the C-terminal helices closer to the respective protomer as in PaSurE. Chapter 3 presents a comparative analysis of the hinges involved in C-terminal helix swapping in PaSurE and StSurE. Based on the comparison of structure and sequence, crucial residues important for C-terminal helix swapping in StSurE were identified as D230 and H234. The chapter describes the construction of mutants obtained by substituting D230 and H234 by alanine and their biophysical characterization. Finally it describes structural studies carried out on these mutants. The mutation H234A and D230A/H234A resulted in highly distorted dimers, although helix swapping was not avoided. Comparative analysis of the X-ray crystal structures of native StSurE and mutants H234A and D230A/H234A reveal large structural changes in the mutants relative to the native structure. However the crystal structures do not provide information on the changes in dynamics of the protein resulting from these mutations. To gain better insights into the dynamics involved in the native and mutants H234A and D230A/H234A, MD simulations were carried on using GROMACS 4.0.7. Chapter 4 deals with a brief description of the theory of molecular dynamics, followed by results of simulation studies carried out on monomeric and dimeric forms of StSurE and dimeric forms of its mutants H234A and D230A/H234A. The conformational changes and dynamics of different swapped segments are discussed. Crystal structures of H234A and D230A/H234A mutants reveal that they form highly distorted dimers with altered dimeric interfaces. Chapter 5 focuses on comparison of dimeric interfaces of the native StSurE and hinge mutants H234A and D230A/H234A. Based on the analysis, three sets of interactions were selected to investigate the importance of the interface formed by swapped segments in StSurE mutants H234A and D230A/H234A. One of the selected sites corresponds to a novel interaction involving tetramerization loop in the hinge mutants H234A and D230A/H234A resulting in a salt bridge between E112 – R179’ and E112’ – H180 (prime denotes residue from the other chain of the dimeric protein). This salt bridge seems to stabilize the distorted dimer. It is shown by structural studies that the loss of this salt bridge due to targeted mutation restores symmetry and dimeric organization of the mutants. Loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE not only leads to large structural changes but also alters the conformation of a loop near the active site. It is of interest to understand functional consequences of these structural changes. StSurE is a phosphatase, and its activity could be conveniently monitored using the synthetic substrate para nitrophenyl phosphate (pNPP) at pH 7 and 25 ºC. Chapter 6 deals with the functional studies carried out with various StSurE mutants. The studies suggest that there is a drastic loss in phosphatase activity in hinge mutants D230A, H234A and D230A/H234A, while in the salt bridge mutants the function seems to have been restored. Few of these mutants also exhibit positive cooperativity, which could probably be due to altered dynamics of domains. Sesbania mosaic virus (SeMV) is a plant virus, belonging to genus sobemovirus. SeMV is a T=3 icosahedral virus (532 symmetry) made up of 180 coat protein (CP) subunits enclosing a positive-sense RNA genome. The asymmetric unit of the icosahedral capsid is composed of chemically identical A, B and C subunits occupying quasi-equivalent environments. Residues 48 – 59 of the N-terminal arms of the C subunits interact at the nearby icosahedral three-fold axes through a network of hydrogen bonds to form a structure called the “β-annulus”. Residues 60 – 73 form the “βA-arm” that connects the N-terminal β-annulus to the rest of the protomer. Various studies on SeMV-CP suggest that different lengths of the N-terminal segments affect the assembly of virus. It might be possible to exploit this flexibility of the N-terminus in SeMV-CP to introduce swapping of this segment between two 2-fold related C subunits as is found in Rice yellow mottle virus (RYMV), another sobemovirus, with which SeMV shares significant sequence similarity. Chapter 7 focuses on attempts made to examine the mutational effects planned to introduce domain swapping. The strategy used for introducing swapping in SeMV-CP was based on the sequence of the βA-arm or the hinge involved in swapping of β-annulus in RYMV. TEM images of the mutant virus like particles obtained suggest that they are heterogeneous. These mutants could not be crystallized, probably due to the heterogeneity. However, the assembly of the expressed proteins to virus like particles was profoundly influenced by the mutations. Chapter 8 discusses various crystallographic, biophysical and biochemical techniques used during these investigations. Finally the thesis concludes with Conclusions and Future perspectives of the various studies reported in the thesis. In summary, I have addressed the importance of amino acid residues and interactions of hinges involved in domain swapping for the quaternary structure and function of proteins.

The PhD thesis is on the studsy of the influence of the ftsZ antisense RNA and FtsH protease on the synthesis and function of the Escherichia coli cytokinetic protein, FtsZ, which mediates septation during cell division. Thus, it involves three molecules, FtsZ, ftsZ antisense RNA, and FtsH protease. While the E. coli ftsZ antisense RNA is being identified and structurally and functionally characterised for the first time, there has been some earlier studies in the laboratory in which the FtsH protease was found to have influence on the presence of the FtsZ rings at the mid-cell site. The Chapter 1 is the Introduction to the thesis presented in 3 parts –Part 1A, 1B, and 1C, introducing FtsZ and bacterial cell division, bacterial antisense RNAs, and FtsH protease, respectively. The Chapter 2 gives the description of the Materials and Methods used in the study. The Chapter 3 presents the identification, structural and functional characterisation of the ftsZ cis-antisense RNA, and its role in the regulation of FtsZ protein levels. Initially, the expression of cis-encoded antisense RNA from E. coli ftsZ loci was demonstrated during the different growth phases of the bacterium (RT-PCR/qPCR data). Antisense RNA is expressed from three promoters (primer extension and promoter probe data) on the complementary strand of the ftsZ coding region and terminates at the singletrand te complementary toftsAthegenethat 3’islocatedregionupstreamof theofftsZ the gene. Induced overexpression of a portion (423 bp) of the antisense RNA, spanning the ftsZ AUG codon and the ribosome binding site of ftsZ mRNA, from pBS(KS) could downregulate the synthesis of FtsZ protein to approximately 30%, leading to cell division arrest and filamentation of the cells at 42°C. This effect was less dramatic at 30ºC, probably due to less melting of the antisense RNA. Immunostaining performed on the induced culture did not show FtsZ ring formation after overnight induction whereas reduction in the proportion of the cells carrying FtsZ rings could be clearly observed after 2 hrs of induction. Real time PCR analysis performed for relative quantitation of ftsZ mRNA and ftsZas RNA from different growth phases (0.2 to 2.5 OD600 nm) showed growth phase dependent expression of the antisense RNA. While the levels of ftsZas RNA were found to be high at lower OD cultures or early growth phase cultures, the levels were found to be low at the late log phase and stationary phase cultures. Thus, when the cells are actively dividing and therefore need more FtsZ, the levels of the ftsZas RNA are high, while the cells are not actively dividing and therefore the FtsZ levels are low, the levels of the ftsZas RNA are low. At any phase of the growth, the ratio of the ftsZ mRNA to the ftsZas RNA was always found to be 6:1. Thus, the physiological role the ftsZas RNA is to maintain the availability of the ftsZ mRNA at a level that is commensurate with the requirement for the FtsZ protein during the different stages of the cell growth and division. The Chapter 4 is on the study of the possible mechanism behind the influence of FtsH protease on the presence of FtsZ rings at the mid-cell site during septation in cell division. Immunostaining for FtsZ in the mid-log phase E. coli cells showed that 82% of the AR3289 (ftsH wild type) cells possessed FtsZ rings, while only 18% of the AR3291 (ftsH-null maintained viable by a suppressor mutation) cells showed Z-rings. While the AR3289 cells showed a cell doubling time of 20 min, the AR3291 cells had a cell doubling time of 45 min. The mass doubling time of AR3289 and AR3291 were 24 min and 54 min, respectively. These distinct differences were found in spite of the suppressor mutation suppressing all the deleterious effects of the lack of the essential protease, FtsH. Complementation of the ftsH-null cells (AR3291) with the wild type FtsH but not with the ATP-binding or ATPase, or protease-defective mutants of FtsH, restored the FtsZ ring status to about 80% of the cells. The growth rate of AR3291 was also partly restored to comparable to that of the wild type cells upon complementation. Western blotting for FtsZ, and the FtsZ-stabilising proteins, FtsA and ZipA, showed that the ftsH-null cells have low levels of FtsA, as compared to those in the isogenic wild type cells (AR3289). The levels of FtsZ and ZipA were comparable in both the cells. Quantitative PCR performed for different cell division genes within the dcw cluster showed no sign of change in the ftsA transcript levels in the ftsH-null cells, suggesting that the low levels of FtsA in the ftsH-null cells were not due to transcriptional downregulation. Further experiments showed that the half-life of FtsA protein in the AR3289 cells was 45 min, while that in the AR3291 cells was 24 min. This experiment showed that the low levels of FtsA in the ftsH-null cells was due to the low half-life of FtsA in the cells. Growth synchronisation of the AR3289 and AR3291 cells showed that the levels of FtsA prior to cell division stage do not increase in the ftsH-null cells as much as in the isogenic wild type cells. Thus, the ftsH-null cells must be somehow managing the division through the partial stabilisation of FtsZ rings by ZipA. Interestingly, immunostaining for FtsH in AR3289 cells showed the presence of FtsH at the mid-cell site, as co-localised with FtsZ, for a brief period prior to cell constriction. These observations suggest the involvement of FtsH in cell division process. The faster degradation of FtsA in the absence of FtsH protease implies that another protein, which may be a protease that directly degrades FtsA or a chaperone that helps the unfolding of FtsA for degradation, might be the substrate of FtsH protease. The absence of FtsH protease brings up the levels of this unknown protein, which in turn facilitates (if it is a chaperone) degradation of or directly degrades (if it is a protease) FtsA. This model for the link among FtsH, FtsA levels, and the presence of FtsZ has been proposed based on the observations. Thus, the present study reveals for the first time an FtsA-linked role for FtsH protease in the presence of FtsZ ring at the mid-cell site and hence in bacterial septal biogenesis. The thesis is concluded with the list of salient findings, publications, and references.

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).