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Structural and Functional Investigations of Selected Hydrolases at Molecular Level

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Structural and Functional Investigations of Selected Hydrolases at Molecular Level Structural and functional investigations of selected hydrolases at molecular level Thesis Submitted to AcSIR For the Award of the Degree of DOCTOR OF PHILOSOPHY in BIOLOGICAL SCIENCES by Ekta Shukla Registration Number: 10BB12A26067 Under the guidance of Dr. Dhanasekaran Shanmugam (Research Supervisor) Dr. Sushama Gaikwad (Research Co-Supervisor) DIVISION OF BIOCHEMICAL SCIENCES CSIR-NATIONAL CHEMICAL LABORATORY PUNE – 411008, INDIA August 2017 Abstract of the thesis Chapter 1: Introduction The importance and updates in protein folding/ unfolding research has been discussed. Significance of studying the structure-function relationship of proteins through various experimental and theoretical approaches has also been presented. Being the largest and most diverse class of enzymes, hydrolases offers an opportunity to explore the conformational/ topological diversity which forms the basis of their differential biological activity. An introduction to hydrolases in terms of their diversity, classification, structure and function is given. The available information and literature survey on selected hydrolases has been summarized as the basis of studies undertaken in the thesis. Chapter 2: Structure-function studies of serine protease from Conidiobolus brefeldianus MTCC 5185 (Cprot) The serine protease from Conidiobolus brefeldianus MTCC 5185 (Cprot) is a monomeric 28 kDa protein showing optimum proteolytic activity at pH 9.0 and 50 °C. Cprot was considered as an interesting candidate for structural and functional studies owing to its commercial applications and unique properties. The results of the chapter are divided into three sections. The first section in the present chapter includes analysis of structural elements of Cprot using different biophysical techniques. The microenvironment of three tryptophans in Cprot was studied using steady state fluorescence and solute quenching studies with neutral (acrylamide) and ionic (I- and Cs+) quenchers. Acrylamide was found to be the most efficient solute for native Cprot, which quenched the fluorescence intensity with −1 Stern-Volmer constant Ksv as 3.9 M . The resistance offered by Cprot towards different proteases and its auto-chewing property was studied by biochemical activity assays. In the second section of the chapter, our understanding of the conformational and functional dynamics of Cprot, in presence of various stress conditions has been discussed. The enzyme was found to be active over a wide pH range, except for extremely acidic Page 3 condition. The thermal denaturation of the enzyme was irreversible and observed above 55 °C, after which both the structure and function of the enzyme were lost. The protease was interestingly, stable in organic solvents up to 50 % (v/v) concentration of alcohols and dimethyl sulfoxide. Alcohols showed α-helix inducing effect on Cprot and its stability in presence of fluorinated alcohols (5-10 %) was also observed. Conformational changes of Cprot during guanidine hydrochloride induced denaturation indicated multi step unfolding of Cprot, involving several intermediates. The melting profile observed for the native Cprot and for the enzyme treated under various stress conditions, correlated well with the corresponding structural and functional transitions obtained. In the last section of the chapter, we intended to construct the 3D model of Cprot. Since, the three dimensional structure of enzymes often provides significant information to understand its active site, secondary structural elements and overall tertiary conformation. Cprot is a β-sheet rich protein as indicated by far-UV CD spectrum and the homology model. The attempt made to identify the sequence of Cprot using C. coronatus proteases revealed its similarity to a trypsin-like protease belonging to PA clan of proteases with His-64, Asp-113 and Ser-208 as putative catalytic triad. The FTIR spectrum of Cprot also resembled with that of trypsin. Chapter 3: Structure-function studies of trehalase from Drosophila melanogaster (DmTre) and Chironomus ramosus (CrTre) Trehalase is a physiologically important glycosidase, known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a cDNA segment, encoding the catalytically active trehalase and its characterization from two dipteran insects, Drosophila melanogaster (DmTre) and Chironomus ramosus (CrTre). The results of the chapter are divided into three sections. The first section includes the molecular cloning of a cDNA segment, encoding the insect trehalase (DmTre and CrTre). The sequences for both DmTre and CrTre has been submitted in GenBank with accession numbers KU049688 and KX857662, respectively. The deduced amino acid sequences were characterized in silico by subjecting them to homology search, multiple sequence alignment and phylogenetic tree construction, revealing their identity to other trehalases which belong to glycoside hydrolase family 37. The instability index and aliphatic index suggested the in vivo- and thermo-stable nature of the protein, respectively. Page 4 The second section of the chapter consists of homology modeling and molecular docking studies, which provide insights on tertiary structure and active site of the enzyme, identifying glutamate and aspartate as the putative catalytic residues. In silico docking of trehalose in the active centre pocket of two trehalases, i.e. E. coli trehalase (prokaryotic) and DmTre (eukaryotic), revealed substantial differences in the binding interactions and affinity of substrate (trehalose) with the enzyme (trehalase). An intrinsic region was also predicted in DmTre and CrTre, which suggested that a small segment between 190 to 215 and 245 to 264 amino acids, respectively, is prone to disorderness. Further, the conserved regions and catalytically important residues were found to be formed majorly of loops, which tend to be evolutionarily conserved. In the third section of the chapter, heterologous expression of DmTre in Escherichia coli has been described using two different vectors viz., pET28a and pCOLD TF and compared for their variable soluble expression, purification and activity of the recombinant enzyme. The recombinant enzyme was also characterized biophysically using far-UV CD and DSF which indicated that trehalase is α-helix rich protein (also evident from the homology model). A novel PPII fold was observed in the far-UV CD scan of the DmTre-inclusion bodies, which is occasionally formed by the unordered structures. The functional stability of DmTre, was then tested against various physicochemical stressors like pH, temperature, denaturants, detergents, organic solvents and proteolytic environment. The enzyme was found to be active over a wide pH range and temperature up to 60 °C, with optimum pH and temperature being 6.0 and 55 °C respectively. Chapter 4: Summary and conclusion Understanding the relationships between protein structure and function at the molecular level, remains a primary focus in structural biology. To understand the structure-function paradigm, useful structural information comes from the primary amino acid sequences and the associated tertiary structures. This chapter discusses the major highlights of the thesis and summarizes the characteristics of the two hydrolases under study. The in vitro and in silico studies described in this thesis contribute to our knowledge of the interplay between the stability, structure and function of the enzymes at molecular level, which can serve as a structural toolbox to improve their efficiency in future. Page 5 Chapter 4 Summary and conclusion Ekta Shukla AcSIR Ph.D. Thesis (2017) 4.1 Introduction Understanding the relationships between protein structure and function at the molecular level, remains a primary focus in structural biology with important consequences in diverse areas such as drug designing, therapeutics and in various industries like textile, agro-based, food and feed etc. To understand the structure-function paradigm, useful structural information comes from the primary amino acid sequences and the associated tertiary structures. Several recent developments in the fields of molecular biology, genetics, biochemistry, protein engineering and bioinformatics have accelerated the research in the "protein universe" (1). Protein structure-function relationships can be investigated by asking how nature has engineered protein structures to perform a variety of functions. In short, there are three elementary assumptions (2): Different structures come from different arrangements of amino acids. If amino acids change, so does the shape which affects function of the protein. Physical and chemical parameters of protein are important in maintaining correct structure and proper function. Thus, full understanding of a molecular system comes from careful examination of the sequence-structure-function triad (Fig. 1). Furthermore, proteins display diverse sequence-structure-function similarity relationships. Usually, proteins with high sequence identity and high structural similarity tend to possess functional similarity and evolutionary relationships; however, examples of proteins deviating from this general relationship of sequence/structure/function homology are well-recognized (table 1). For example, high sequence identity but low structure similarity can occur due to conformational plasticity, mutations, solvent effects, and ligand binding (3, 4, 5). Therefore, the major challenges in structural biology are: Below 30 % protein sequence identity
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