Publications (43) View all
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Article: C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism.
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ABSTRACT: The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism.FEBS Journal 06/2011; 278(17):3012-24. · 3.79 Impact Factor -
Article: Role of remote scaffolding residues in the inhibitory loop pre-organisation, flexibility, rigidification and enzyme inhibition of serine protease inhibitors ☆
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ABSTRACT: residues like Arginines or Asparagine shape-up the inhibitory loop and favor the resynthesis of cleaved scissile bond. However, role of remote scaffolding residues, which are not involved in religation, was not properly explored. Crystal structures of two engineered Winged bean chymotrypsin inhibitor (WCI) complexed with Bovine trypsin (BPT) namely L65R-WCI:BPT and F64Y/L65R-WCI:BPT show that the inhibitory loop of these engineered inhibitors are recognized and rigidified properly at the enzyme active site like other strong trypsin inhibitors. Chimeric protein ETIL-WCIS, having loop of Erythrina caffra Trypsin Inhibitor, ETI on the scaffold of WCI, was previously shown to behave like substrate. Non-canonical structure of the inhibitory loop and its flexibility are attributed to the presence of smaller scaffolding residues which can not act as barrier to the inhibitory loop like in ETI. Double mutant A76R/L115Y-(ETIL-WCIS), where the barrier is reintroduced on ETIL-WCIS, shows regaining of inhibitory activity. The structure of A76R/L115Y-(ETIL-WCIS) along with L65R-WCI:BPT and F64Y/L65R-WCI:BPT demonstrate here that the lost canonical conformation of the inhibitory loop is fully restored and loop flexibility is dramatically reduced. Therefore, residues at the inhibitory loop interact with the enzyme playing primary role in recognition and binding but scaffolding residues having no direct interaction with the enzyme are crucial for rigidification event and the inhibitory potency. B-factor analysis indicates that the amount of inhibitory loop rigidification varies between different inhibitor families.BBA PROTEINS AND PROTEOMICS. 05/2012; -
Article: Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitors.
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ABSTRACT: For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed "mini-proteins," containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop-scaffold compatibility, we prepared three chimeric proteins ECI(L)-WCI(S), ETI(L)-WCI(S), and STI(L)-WCI(S), where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECI(L)-WCI(S) and STI(L)-WCI(S) behave like good inhibitors, ETI(L)-WCI(S) behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETI(L)-WCI(S) shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETI(L)-WCI(S) leading to a loss of canonical conformation, explaining its substrate-like behavior. Incorporation of this barrier back in ETI(L)-WCI(S) through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop-scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design.Protein Science 03/2010; 19(3):593-602. · 2.80 Impact Factor -
Article: Production and recovery of recombinant propapain with high yield.
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ABSTRACT: Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a(+) each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble His-tagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400mg of properly refolded propapain was obtained from 1L of bacterial culture. The expressed protein was further verified by Western blot analysis by cross-reacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease.Phytochemistry 03/2009; 70(4):465-72. · 3.35 Impact Factor -
Article: Kristall‐ und Molekülstruktur von 4‐Amino‐1‐methyl‐2‐(methylthio)pyrimidinium‐chlorid — Ein Modell für S‐alkyliertes 2‐Thiocytidin
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ABSTRACT: 4-Amino-1-methyl-2-(methylthio)pyrimidinium-chlorid (3) kristallisiert in der triklinen Raumgruppe P1 mit zwei Molekülen in der asymmetrischen Einheit. Die Lösung der Struktur durch direkte Methoden führte nach Verfeinerung zu einem R-Faktor von 0.054. Die Moleküle sind planar mit den S-Methyl-Gruppen in cis-Stellung zur C(2)–N(3)-Bindung. Bindungsabstände und -winkel der beiden Moleküle sind innerhalb der Standardabweichungen gleich und deuten auf weitgehend aromatische Konstitutionen. Im Kristallgitter bildet eines der beiden Chlorid-Ionen in der asymmetrischen Einheit drei, das andere dagegen nur eine Wasserstoffbrücken-bindung aus.Crystal and Molecular Structure of 4-Amino-1-methyl-2-(methylthio)pyrimidinium Chloride – A Model for S-Alkylated 2-ThiocytidineCrystals of 4-amino-1-methyl-2-(methylthio)pyrimidinium chloride (3) are triclinic, space group P1, with two molecules in the asymmetric unit. The structure was solved by direct methods and refined to R = 0.054. The molecules are planar with the S-methyl group in a cis-relationship with the C(2)–N(3)-bond. Bond lengths and angles for the two molecules are equal within standard error limits, and indicate largely aromatic structures. In the crystal matrix, one of the two chloride ions in the asymmetric unit forms three hydrogen-bonds, the other only one.Berichte der deutschen chemischen Gesellschaft 01/2006; 110(1):353 - 360. · 2.94 Impact Factor
