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Authors: Singh, Anamika
Keywords: Proteins
biophysical techniques
macromolecular X-ray
nuclear magnetic resonance
Issue Date: Jul-2015
Abstract: Proteins are requisite to life as we know it. Proteins are polymers constructed from 20 different amino acids. Proteins execute their functions due to their capability to bind to diverse macromolecules distinctively and ability to form different strong interactions with them. They are involved in virtually all cell functions. Along with their many important roles, they provide structure (e.g. collagen), transport (e.g. haemoglobin) and catalytic power (e.g. catalase) for biological systems and are fine-tuned to be highly specific for their function. It is well established that for all proteins, both their structure and dynamics are intimately tied to their function. Thus protein structure and dynamics are fundamental components for building our foundation of biochemistry. Thus the field of structural biology is broad to understand the structure-function relationships of proteins. Two powerful tools to examine structure-function relationships are the harmonizing biophysical techniques of macromolecular X-ray crystallography and nuclear magnetic resonance (NMR). X-ray crystallography provides atomic resolution structures that allow us to see well-defined positions of atoms. These “pictures” show us what a protein’s structure looks like. While NMR can be used to solve protein structure, NMR’s strength is in its ability to probe enzyme motions (dynamics) and changes in local environment with per-residue resolution. These two techniques, along with many other macromolecular biophysics tools (e.g. fluorescence, circular dichroism, mass spectrometry, isothermal titration calorimetry, surface Plasmon resonance) provide complementary information that helps to create a detailed picture of a protein’s structure and dynamics that can be interpreted to understand its function. Nearly 2.7 billion years ago, the introduction of molecular oxygen (O2) into our atmosphere, resulted in the reactive oxygen species (ROS) as unwelcome companions in the ecosystem. Although they control many different processes in plants, their toxic nature is also capable of injuring cells. Oxidative stress is an uncompleted battle between highly reactive free radicals and the systems designed to soften their effects. When free radicals are winning this battle, oxidative damage occurs. ROS react with cell biomolecules, leading to organelle dysfunction. Oxidative stress may be seen to start at the molecular level, with a direct interaction between free radicals and a protein, a lipid, carbohydrates, or nucleic acids. Indeed certain diseases results because of failure to respond to these damaging consequences, causing damage both locally or systemically. ii Oxidative stress is a foremost problem for whichever organisms that uses oxygen as an electron acceptor. Because, as incomplete reduction of oxygen to water can yield reactive oxygen species (ROS) such as the superoxide anion (O2 -), hydrogen peroxide (H2O2), and the hydroxyl radical (•HO) ions. Build up of these highly reactive species can lead to damage to proteins, nucleic acids, and membranes. ROS are also produced by the immune system to kill invading microbes. Therefore, an ability to combat these compounds is a key to the survival of bacterial pathogens in the environment and the host exerted via their antioxidant systems. In the present work, proteins of antioxidant system were cloned purified and characterized from genomic DNA of Candidatus Liberibacter asiaticus (CLA) which includes Bacterioferritin comigratory protein; 1-Cys Prxs and thioredoxin. These proteins revealed multiple functions which may have strong impact in relation to its biotechnology applications. The CLa-BCP characterized from CLA showed DNA-binding, in-vitro antioxidant and peroxidase activities against varied peroxide substrates. The cloning of gene revealed a 471bp bp ORF encoding a polypeptide of 157 amino acid residues which included the N-terminal his-tagged with a calculated molecular mass of 19.670 kDa. The protein was cloned in pETTEV expression vector. The variant form of CLa-BCP after introduction of non-conserved cysteine at 77th position, CLa-BCPS77C by site directed mutagenesis also purified and characterized. It also exhibited DNA-binding, in-vitro antioxidant and peroxidase activities against varied peroxide substrates. The thesis has been divided into three chapters. Chapter 1 reviews the literature describing about plant defense against microbes’ invasion which summarizes the reactive oxygen species (ROS), reactive nitrogen species (RNS) network and counter antioxidant network prevalent in microorganism to combat the oxidative stress. An array of antioxidant machinery present in microorganism to counteract the deleterious effect of ROS includes non-enzymatic and enzymatic antioxidants. Enzymatic antioxidants include superoxide dismutase, catalase, cytochrome c peroxidases, and alkyl hydroperoxide-reductase (AhpC). BCPs (Bacterioferritin comigartory proteins) are least characterized subfamily of (AhpC) Prx family which is also played an important role in detoxifying hydroperoxides. Chapter 2 describes the cloning, expression, purification of 1-Cys Prxs CLa-BCP and its biochemical characterization along with the cloning of its reductant partner CLa-TrxA. The cloning and sequence analysis revealed that CLa-BCP is 1-cys Prx protein having single peroxidatic key cysteine residue (CpSH) without resolving cysteine (CRSH). The purification of CLa-BCP and its variant CLa-BCPS77C was accomplished using two step purification iii method employing Ni-NTA affinity and size exclusion chromatography. The purified protein showed a single band in both and non-reducing condition depicting the predominance of monomers of 19.6 kDa. The purified CLa-BCP when applied to size exclusion chromatography column showed a single peak corresponding to dimeric species. Gel filtration describes its concentration dpendent oligomeric nature; possess dimers with predominant monomeric species. An introduction of cysteine residue replacing 77th serine residue in α3 helix (CLa-BCPS77C) results into intermolecular disulfide bonds revealing its dimeric nature. The reductant partner Thioredoxin (CLa-TrxA) was successfully cloned, expressed and purified to its homogeneity. The SDS-PAGE analysis showed that it forms dimer but predominantly monomeric in nature. The purified protein exhibited peroxiredoxin, DNAbinding and in-vitro antioxidant activities also. Both showed a strong ROS scavenging activity towards breast cancer (MCF-7) and mouse mesenchymal stem cells (C3H10t1/2) cell lines. The cell viability was estimated by MTT assay towards Adenocarcinoma breast cancer cells (MCF7), Fibroblast-like cell line (COS7) indicated the protective effect of the protein against hydrogen peroxide mediated cell killing. Both CLa-BCP and CLa-BCPS77C showed DNA-binding capability resulting in protection from oxidative nicking of supercoiled DNA. Chapter 3 describes the biophysical characterization of CLa-BCP and CLa-BCPS77C, preliminary crystallization of CLa-BCP and in-silico analysis regarding its versatility in function. Secondary structure analysis by CD showed that both proteins are quite ordered in its reduced form. CD at different temperature showed that CLa-BCP quite steady even at higher temperature like 90°C and maintains its β sheet structure. Intrinsic fluorescence studies at different pH demonstrated the conformational stability of protein. During crystallization, initially CLa-BCP crystals were poor-quality inter grown sea urchin like crystals. Addition of low percentage agarose results into well ordered crystals. While mounting crystals, crystals when kept in low ethylene glycol followed by annealing results into better diffraction. Thus for better diffraction of BCP crystals perquisite were very low percentage of ethylene glycol and annealing. The frozen crystal diffracted X-rays to 2.4 Å resolution using the Cu rotatinganode generator. Analysis of crystal symmetry and recorded diffraction patterns specified that the CLa-BCP crystal belongs to monoclinic space group type C2 having unit-cell parameters a= 92.226, b= 151.9440, c= 139.4940 A °, α= γ=90°, β= 108.7140. The amino acid sequence comparisons and phylogenetic analysis of CLa-BCP showed significant homology to BCPs family proteins (both 1-Cys and 2-Cys) from different pathogenic bacterium but higher similarity with bacterial AhpCs. Model has been built on the basis of amino acid sequence that substantiates its α/β structure predominantly β structure and sites for catalysis. iv In summary, our studies confirm that peroxiredoxins; Prxs family proteins are multifaceted proteins which not only involve in detoxification of peroxides but potent scavenger of ROS precluding DNA from oxidative damage. Many proteins of this family have been shown to possess more than one function which includes redox regulation, antioxidant defence and signalling of different organisms. The proposed work will certainly enhance our understanding in terms of specificity and mechanism of action of these antioxidant enzymes from CLA and will lead to the development of effective inhibitor molecules to control citrus greening as well as contribute towards understanding the significance of free radical scavenging therapy for several diseases.
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