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Colin Berry - One of the best experts on this subject based on the ideXlab platform.
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generation of an affinity matrix useful in the purification of natural Inhibitors of plasmepsin ii an antimalarial drug target
Biotechnology and Applied Biochemistry, 2009Co-Authors: Angel Ramirez, Judith Mendiola, Yasel Guerra, Anabel Otero, Aida Hernandezzanui, Beatriz Garcia, Colin Berry, Maria A ChavezAbstract:: An affinity matrix containing the antimalarial drug target Plm II (plasmepsin II) as ligand was generated. This enzyme belongs to the family of Plasmodium (malarial parasite) Aspartic Proteinases, known as Plms (plasmepsins). The procedure established to obtain the support has two steps: the immobilization of the recombinant proenzyme of Plm II to NHS (N-hydroxysuccinimide)-activated Sepharose and the activation of the immobilized enzyme by incubation at pH 4.4 and 37 degrees C. The coupling reaction resulted in a high percentage immobilization (95.5%), and the matrices obtained had an average of 4.3 mg of protein/ml of gel. The activated matrices, but not the inactive ones, were able to hydrolyse two different chromogenic peptide substrates and haemoglobin. This ability was completely blocked by the addition of the general Aspartic-Proteinase Inhibitor, pepstatin A, to the reaction mixture. The matrices were useful in the affinity purification of the Plm II Inhibitory activity detected in marine invertebrates, such as Xestospongia muta (giant barrel sponge) and the gorgonian (sea-fan coral) Plexaura homomalla (black sea rod), with increases of 10.2- and 5.9-fold in the specific Inhibitory activity respectively. The preliminary K(i) values obtained, 46.4 nM (X. muta) and 1.9 nM (P. homomalla), and the concave shapes of the inhibition curves reveal that molecules are reversible tight-binding Inhibitors of Plm II. These results validated the use of the affinity matrix for the purification of Plm II Inhibitors from complex mixtures and established the presence of Plm II Inhibitors in some marine invertebrates.
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N-terminal extension of the yeast IA3 Aspartic Proteinase Inhibitor relaxes the strict intrinsic selectivity.
The FEBS journal, 2007Co-Authors: Tim J Winterburn, David Wyatt, Lowri H Phylip, Colin Berry, Daniel Bur, John KayAbstract:Yeast IA3 Aspartic Proteinase Inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even Aspartic Proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia Proteinase was selected as the target for initial attempts to engineer IA3 to re-design the specificity. The IA3 polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)7MQ sequence generated Inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA3 concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA3 polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA3 polypeptides that display little affinity for the Pichia enzyme into potent Inhibitors of this Proteinase and thus broaden the target selectivity of this remarkable small protein.
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adaptation of the behaviour of an Aspartic Proteinase Inhibitor by relocation of a lysine residue by one helical turn
Biological Chemistry, 2006Co-Authors: Tim J Winterburn, David Wyatt, Lowri H Phylip, Colin BerryAbstract:In addition to self-inhibition of Aspartic Proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target Aspartic Proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-Proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur.
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the Aspartic Proteinase from the rodent parasite plasmodium berghei as a potential model for plasmepsins from the human malaria parasite plasmodium falciparum
FEBS Letters, 1999Co-Authors: Michelle J Humphreys, Sylvia D Fowler, Annette Klinder, Richard P. Moon, Katharina Rupp, Robert G Ridley, Colin BerryAbstract:The gene encoding an Aspartic Proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general Aspartic Proteinase Inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin Inhibitors is discussed.
Arthur S. Edison - One of the best experts on this subject based on the ideXlab platform.
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ia3 an Aspartic Proteinase Inhibitor from saccharomyces cerevisiae is intrinsically unstructured in solution
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113−117]. Surprisingly, only residues 2−32 of IA3 are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA3 is unstructured in the absence of YprA. Specifically, IA3 produced a CD spectrum characteristic of an unstructured peptide, and the 15N HSQC NMR spectra of IA3 were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA3 by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA3 protein by NMR and comparing the chemical...
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IA3, an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phyl...
Lowri H Phylip - One of the best experts on this subject based on the ideXlab platform.
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N-terminal extension of the yeast IA3 Aspartic Proteinase Inhibitor relaxes the strict intrinsic selectivity.
The FEBS journal, 2007Co-Authors: Tim J Winterburn, David Wyatt, Lowri H Phylip, Colin Berry, Daniel Bur, John KayAbstract:Yeast IA3 Aspartic Proteinase Inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even Aspartic Proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia Proteinase was selected as the target for initial attempts to engineer IA3 to re-design the specificity. The IA3 polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)7MQ sequence generated Inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA3 concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA3 polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA3 polypeptides that display little affinity for the Pichia enzyme into potent Inhibitors of this Proteinase and thus broaden the target selectivity of this remarkable small protein.
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adaptation of the behaviour of an Aspartic Proteinase Inhibitor by relocation of a lysine residue by one helical turn
Biological Chemistry, 2006Co-Authors: Tim J Winterburn, David Wyatt, Lowri H Phylip, Colin BerryAbstract:In addition to self-inhibition of Aspartic Proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target Aspartic Proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-Proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur.
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ia3 an Aspartic Proteinase Inhibitor from saccharomyces cerevisiae is intrinsically unstructured in solution
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113−117]. Surprisingly, only residues 2−32 of IA3 are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA3 is unstructured in the absence of YprA. Specifically, IA3 produced a CD spectrum characteristic of an unstructured peptide, and the 15N HSQC NMR spectra of IA3 were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA3 by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA3 protein by NMR and comparing the chemical...
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IA3, an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phyl...
Ben M Dunn - One of the best experts on this subject based on the ideXlab platform.
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ia3 an Aspartic Proteinase Inhibitor from saccharomyces cerevisiae is intrinsically unstructured in solution
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113−117]. Surprisingly, only residues 2−32 of IA3 are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA3 is unstructured in the absence of YprA. Specifically, IA3 produced a CD spectrum characteristic of an unstructured peptide, and the 15N HSQC NMR spectra of IA3 were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA3 by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA3 protein by NMR and comparing the chemical...
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IA3, an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phyl...
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x ray crystallographic studies of the complex between porcine pepsin and the Aspartic Proteinase Inhibitor pi 3 from the nematode ascaris suum
Advances in Experimental Medicine and Biology, 1998Co-Authors: Jens Petersen, Ben M Dunn, Maia M Chernaia, Chetana Raonaik, Jeffrey L Zalatoris, Michael N G JamesAbstract:Ascaris suum is an intestinal endoparasitic nematode found in pigs. In order to avoid proteolytic digestion by the host the nematode has developed a large battery of Proteinase Inhibitors. Among those is the Aspartic Proteinase Inhibitor PI-3. PI-3 consists of a single polypeptide chain containing 149 residues with a molecular weight of 16.4 kD. It has three disulfide bridges. CD and secondary structure predictions indicate that the fold of PI-3 is mixed alpha/beta.1 Until now only a very few larger Aspartic Proteinase Inhibitors have been characterized. Aspartic Proteinase Inhibitors from the Filarie family of parasites have been reported (Dirofilaria, Brugia and Onchocerca). These Inhibitors are more closely related to each other (~60% sequence identity) than PI-3 (less than 20%).2 The Inhibitor form Ascaris suum show a broad specificity towards members from the pepsin related family of Aspartic Proteinases having Ki’s in the range of 0.2—15 nM (porcine and human pepsin and cathepsin E). Cathepsin D however, is not inhibited by PI-3, although it shares up to 45 identity to the above mentioned Aspartic Proteinases.3 This has important aspects as it might be used to distinguish between cathepsin D and E. The following report describes the progress in the structure determination of the molecular complex between porcine pepsin and PI-3.
Terry B Green - One of the best experts on this subject based on the ideXlab platform.
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ia3 an Aspartic Proteinase Inhibitor from saccharomyces cerevisiae is intrinsically unstructured in solution
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113−117]. Surprisingly, only residues 2−32 of IA3 are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA3 is unstructured in the absence of YprA. Specifically, IA3 produced a CD spectrum characteristic of an unstructured peptide, and the 15N HSQC NMR spectra of IA3 were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA3 by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA3 protein by NMR and comparing the chemical...
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IA3, an Aspartic Proteinase Inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution.
Biochemistry, 2004Co-Authors: Terry B Green, Omjoy K Ganesh, Stephen J Hagen, Lowri H Phylip, Ben M Dunn, Kyle Perry, Leif Smith, Timothy M Logan, Arthur S. EdisonAbstract:IA3 is a highly specific and potent 68-amino acid endogenous Inhibitor of yeast Proteinase A (YprA), and X-ray crystallographic studies have shown that IA3 binds to YprA as an α-helix [Li, M., Phyl...
