Characterization of proteolytic machinary of pyrobaculum calidifontis in vitro vs in silico
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Date
2016
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UMT, Lhr
Abstract
Protease and peptidase are imperative constituents for every complex living entity and play crucial roles insides and outside the cell. They are paramount for development and even survival of any organism. They play a vital role in foraging for proteins thus having a special alliance with proteolysis and cellular roles. The present dissertation is focused on analysis of the proteolytic activity of such a few proteases. The exposure of high temperature to proteins denature it and ends its fuction while the special behavior of proteins from Archaea that resists such extreme conditions enables us to work on its protease for observing different tasks of protease including the distinctive proteolysis. Their special outer membranous structure with isoprene unit and saturated fatty acids makes them a great area of interest. . For this purpose, the Archaeal specie Pyrobaculum calidifontis was chosen. The organism is facultative aerobic obligate hyperthermophilic archaeon. Three open reading frames, identifying Pcal_0773, Pcal_0842 and Pcal_0970, were selected from genome and primers were designed for their gene amplification using PCR and agarose gel electrophoresis and sequenced. The Pcal_0773 which is prolyl oligopeptidase was found to have an amplified size of 1.6kb while that of Pcal_0842 as glutamyl aminopeptidase was 1.12kb and Pcal_0970 as glycosylasparaginase precursor protease was 914b only. The homology modelling predict the 3D structure, protein blast was performed followed by docking with different substrates and ligands too find their binding in order to minimize the prolonged laboratory work. The predicted molccular model of prolyl oligopeptidase (Pcal_0773) have 13 α-helices and 35 β-strands and found to have β-propeller domain which confirmed it to be prolyl
oligopeptidase with seven blades which are formed by the clusters of β-sheets. The protein blast gave maximum identity of 33% with Aeropyrum pernix and
Stenotrophomonas maltophilia. The multiple alignment sequences also showed the conserved and important Asparagine residue which is important during the formation of oxyanion hole during proteolytic activity. Moreover, it was docked with GLY-PHEARG-PRO and SUC-GLY-PRO substrate for confirmation of binding sites while docking with Z-PRO-PROLINAL inhibitor proved the presence of catalytic triad which is Ser431, His545 and Asp513. The predicted molecular model of Pcal_0842 which is glutamyl aminopeptidase was predicted by homology modelling which was very managed and orderly having 13 αhelices and 22 β-strands. The protein blast and phylogenetic tree gave a similarity with protease from Desulfurococcus kamchatkensis with percentage of 51% while aligned sequences showed conserved active site. The docking was performed with glycerol ligand which expressed its binding site to be Glu208. The docking with GLU-ALA, ASP-ALA and SER-ALA substrate defined its catalytic site to be Glu208 residue while the inhibitor wasd also predicted by this process. Hence the Thiol derivatives of glutamate and aspartate were docked as inhibitor to figure out their impact on catalytic site. The molecular structure glycosylasparaginase precursor peptidase (Pcal_0970) was made by homology modelling. The protein blast and phylogenetic analysis proposed its closed evolutionary relation with Escherichia coli glycosylasparaginase precursor
peptidase with 49% while aligned sequences disclosed the conserved Thr residue at different sites. The docking with NAG (N-acetyl-glucosaminidase) and BMA (Beta-
mannose) substrates was performed to analyse the autoproteolytic activity of protease along with the activity of conserved Asp165 residue. The docking was also performed with aspartic acid and glycine which act as inhibitor to express the binding site at Thr169 as well as Asp165. The catalytic site on substrate protein was introduced with high accuracy rate and convenience. The properly folded proteins were analyzed having most hydrophilic residues exposed and mostly hydrophobic residues buried. This fact compelled to find relative solvent accessibility (RSA) as solvent accessibility that plays a key role in prediction of proteolysis. The solvent accessibility was evaluated by finding the exposed and buried residues in protein and their availability. The hydrophobicity was also analyzed and it was considered that the exposed bulky groups may hinder proteolysis.The system was designed that automatically conveys the information of all the buried and exposed residues in short time and with high accuracy of about 95%. The accessibility and availability of residues were analyzed and proteolytic cleavage mechanism was applied on it computationally. Thus the proteolysis by almost all the protease in P. calidifontis was predicted very efficiently and within very less time of 0.02 milliseconds per protease. In this thesis, the molecular structures of three proteases i.e. Pcal_0773, Pcal_0842, Pcal_0970, were successfully predicted using homology modelling. The docking process showed the binding of ligands, substrates and inhibitors which confirmed its active sites. The software was designed to analyze the proteolytic activity of all possible protease in P. calidifontis.