Kumar, Kancherla Aswani
The work presented in this thesis describes the determination of structures of peptides and proteins at atomic resolution. Nuclear Magnetic Resonance (NMR) spectroscopy was used as the principal method of investigation. The thesis is divided into three parts. Part I of the thesis consists of chapters 1 to 4, and deals with structural studies of two novel conotoxins. Part II of the thesis consists of chapter 5 and deals with structural studies of Small MutS Related (Smr) domain from Helicobacter pylori MutS2. Part III of the thesis consists of Appendices A to D. Appendix A describes implementation of a novel...
Under hostile conditions, bacteria elicit stress response. Such stress response is regulated by a secondary messenger called (p)ppGpp. (p)ppGpp is involved in wide range of functions such as GTP homeostasis, biofilm formation and cell growth. Its regulation and mode of action is not well understood. This work has been initiated with an aim to gain insights into the molecular basis of stress response. (p)ppGpp was discovered on the chromatogram of cell extract from starved E. coli cells. (p)ppGpp is synthesized and hydrolyzed by Rel/SpoT in Gram negative bacteria (such as E. coli), and by bifunctional enzyme called Rel in Gram...
A major challenge in biology is to understand and predict the effect of mutations on protein structure, stability and function. Chapter 1 provides a general introduction on protein sequence-structure relationships and use of the CcdAB toxin-antitoxin system as a model to study molecular determinants of mutant phenotypes. In Chapter 2, we describe the use of saturation mutagenesis combined with deep sequencing to determine phenotypes for 1664 single-site mutants of the E. coli cytotoxin, CcdB. We examined multiple expression levels, effects of multiple chaperones and proteases and employed extensive in vitro characterization to understand how mutations affect these phenotypes. While general...
Assembly of virus capsid protein (CP) into icosahedrally symmetric particles is an intriguing and elegant process. In most cases of virus assembly, a large number of identical protein subunits self-assemble to generate a shell that protects the viral genome. Studies on virus assembly have resulted in a new scientific technique that uses these proteinaceous shells as nano-particles for a variety of biological applications. The current thesis deals with understanding the factors that govern the assembly of the Sesbania mosaic virus (SeMV) and a pleomorphic virus, Tobacco streak virus (TSV).
CP of SeMV, a T=3 plant virus, consists of a disordered...
Conserved residues in protein are crucial for maintaining structure and function, either by direct involvement in chemistry or indirectly, by being essential for folding, stability and oligomerisation and are mostly clustered near active sites. The variability of sequences of the same protein from diverse organisms is a reflection of the selective pressures of evolution.
Sequence conservation analysis with 3397 bacterial triosephosphate isomerase (TIM) sequences using Plasmodium falciparum (Pf) TIM as template, showed full conservation of ten residues, K12, T75, H95, E97, C126, E165, P166, G209, G210 and G228. The integrity of the enzyme active site, which lies near the dimer...
Low intensity and low frequency ultrasound has been shown to modulate ion channel currents, membrane capacitive currents, and as a result, neuronal activity. Ultrasound has been used as a non-invasive way to modulate neuronal activity in vivo using mice as well as human subjects. Ultrasound with acoustic frequency as low as 0.35 MHz can be focussed on a region as small as 2 mm with reversible effects and no increase in temperature. In this study, two ultrasound transducers with different resonant frequency have been used to excite neuronal cultures. The resulting changes in the network properties such as synchronised network...
A cell is a highly complex, ordered, and above all, a robust system. It copes with in-trennel and external uncertainties like heterogeneous stimuli, errors in processing and execution, and changes within and outside the cell. Maintenance of such a system critically depends on a large body of signalling networks and associated regulatory mechanisms. Of the recurrent manoeuvres in cell signalling, protein phosphorylation is the most prominent, and is used as a switch to transmit information and effect-ate various outcomes. It is estimated that 30% of the entire proteome of a typical eukaryotic cell is phosphorylated at one time or another,...
Transcription is a major step in expression of genes of a given organism. Due to environmental constrains this step must be regulated in the favour of the sustenance and growth of the organism. Here comes the relevance of transcription factors, mostly proteins which regulate transcription. One such important group of transcription factors is the zinc finger proteins.
It is well known that in eukaryotes the C2H2 zinc finger domain containing proteins are the largest group of transcription factors while in prokaryotes the largest group of transcription factors are represented by helix-turn-helix motif containing proteins. Till now only two C2H2 zinc...
Regulation of transcription in prokaryotes is primarily governed at the transcription initiation step. This feature has been extensively characterized in model prokaryotes notably Escherichia coli and Bacillus subtilis. Transcription initiation was initially thought to be governed primarily by initiation factors that recruit the RNA polymerase (RNAP) enzyme to initiate expression of given gene. Recent studies reveal multiple mechanisms at play including additional protein factors that can modulate gene expression. Nonetheless, understanding transcription factors is key to rationalize the nuanced changes in prokaryotic gene expression in response to diverse environmental stimuli. This is particularly relevant in the case of the human...
Aditya Kumar, *
Transcription initiation is an important step in the process of gene regulation in prokaryotes. Promoters are stretches of DNA sequence that are present in the upstream region of transcription start sites (TSSs), where RNA polymerase and other transcription factors bind to initiate transcription. Recent advancement in sequencing technologies has resulted in huge amount of raw data in the form of whole genome sequences. This sequence data has to be annotated, in order to identify coding, non-coding and regulatory regions. Computational tools are useful for a quick and fairly reliable annotation of many genome sequences. Promoter prediction is an important step...
The biosynthetic pathway for the production of the dipeptide antibiotic bacilysin has been the subject of intense research over the past three decades. These studies revealed the role of multiple enzymes in the biosynthesis of this antibiotic. The identification of different enzymes was initially guided by genetic studies on different strains of Bacillus. The functional role of some these enzymes have been validated in vitro in the recent past. Despite this, the in vitro synthesis of bacilysin still remains elusive. The focus of this study was on two oxidoreductases - BacC and BacG. In the course of these studies, several...
Yamuna Kalyani, M
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...
A number of viral diseases such as Hepatitis B, small pox, measles, rubella and polio have effective vaccines to control or eradicate them. HIV-1 is a lentivirus which infects human immune cells and leads to the disease called AIDS (Acquired Immuno Deficiency Syndrome). Despite much effort since the three decades of its discovery, there is no effective vaccine against HIV-1. The envelope glycoprotein of HIV-1 is the most accessible protein on the virion surface and is essential for HIV-1 infection. Thus, this protein is the primary target for HIV-1 vaccine design. However, HIV-1 has acquired numerous immune evasive mechanisms to...
Small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones that play a vital role in maintaining protein homeostasis in cells. They are the first line of defence against the detrimental effects of cellular stress conditions like fluctuations in temperature, pH, oxidative and osmotic potentials, heavy metal toxicity, drought and anoxia. Many sHSPs are also constitutively expressed during developmental stages of different plant tissues.
Members of this family are ATP-independent chaperones, with monomeric masses varying from 12-40 kDa. A characteristic feature of sHSPs is their ability to assemble into large oligomers, ranging from dimers to 48-mers. Under stress...
Molecular chaperones are proteins that interact with and aid in stabilization and activation of other proteins. Chaperones help proteins attain their three dimensional conformation, without forming a part of the final structure. Many of the chaperones are stress proteins known as Heat shock proteins (Hsps). Their expression is upregulated in response to various kinds of stress such as heat stress, oxidative stress etc., which threaten the protein homeostasis, by structurally destabilizing cellular proteins, and increasing the concentration of aggregation-prone folding intermediates. The Hsps are classified according to their molecular weight into Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and the small Hsp...
This thesis is concerned with the first ever X-ray crystallographic and complimentary solution studies on mycobacterial lectins. Lectins, described as multivalent carbohydrate binding proteins of non-immune origin, are found in all kingdoms of life. As explained in the introductory chapter, those from plants and animals are the best characterized in terms of structure and function. Although not that extensive, important studies have been carried out on viral, fungal and parasite lectins as well. Bacterial lectins studied so far can be classified in to fimbrial, surface and secretory (or toxic). Applications of lectins include blood typing, cell separation and purification of...
The subiculum is a structure that forms a bridge between the hippocampus and the entorhinal cortex (EC) in the brain, and plays a major role in the memory consolidation process. It consists of different types of pyramidal neurons. Based on their firing behavior, these excitatory neurons are classified into strong burst firing (SBF), weak burst firing (WBF) and regular firing (RF) neurons. In the first part of the work, morphological differences in the different neuronal subtypes was explored by biocytin staining after classifying the neurons based on the differences in electrophysiological properties. Detailed morphological properties of these three neuronal subtypes...
The three-dimensional structures of biological macromolecules and molecular assemblies are becoming increasingly important with the changing methodologies of drug discovery. The structures aid in understanding of protein function at the molecular level: be it a macromolecular assembly, a cytosolic enzyme or an intermembrane receptor molecule. X-ray crystallography is the most powerful technique to obtain the three-dimensional structures of such molecules at or near atomic resolution. With such a wide-spread importance, crystallography is an integral part of structural biology and also of the current drug discovery programs.
The present thesis mainly deals with application of the crystallographic techniques for understanding the...
Since, its discovery over three decades ago, HIV has wrecked havoc worldwide. According to the UNAIDS report 2011, at present 34 million people is living with HIV and AIDS vaccine with broadly neutralizing activity still remains elusive. The envelope glycoproteins on the virion surface, is the most accessible component to the host immune system and therefore is targeted for vaccine design. However, the virus has employed various strategies to avoid the host immune response. The extremely high rate of mutations, extensive glycosylation of the envelope glycoprotein, conformational flexibility of the envelope, has made all the efforts aimed to design a...