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BBA - Proteins and Proteomics (v.1764, #6)
Hybrid spectrin type repeats produced by exon-skipping in dystrophin by Nick Menhart (pp. 993-999).
Dystrophin is the protein whose defect underlies Duchenne Muscular Dystrophy, DMD, a common (1:3500 male births) and fatal condition in which muscle tissue deteriorates leading to death in the second or third decade of life. Dystrophin is coded for by the largest human gene, and one of the most complex. It is translated from at least 7 distinct promoters, with the largest transcripts (which are the ones involved in DMD) containing 79 exons over >2.5 Mbp [K.F. O'Brien, L.M. Kunkel, Dystrophin and muscular dystrophy: past, present, and future, Mol. Genet. Metab. 74 (2001) 75–88, H.M. Sadoulet-Puccio, L.M. Kunkel, Dystrophin and its isoforms, Brain Pathol. 6 (1996) 25–35]. Exacerbating this complexity, it has recently been shown that dystrophin is subject to extensive alternative RNA processing, potentially producing a wide variety dystrophin variants [M. Sironi, R. Cagliani, U. Pozzoli, A. Bardoni, G.P. Comi, R. Giorda, N. Bresolin, The dystrophin gene is alternatively spliced throughout its coding sequence FEBS Lett 517 (2002) 163–166]. The structure of the dystrophin protein is highly modular, with the most common module being a motif termed the spectrin type repeat, or STR, of which there are 24. Each STR is roughly coded for by two exons, and the most common type of multiple exon-skipping events start and end at introns in the middle of STRs [R.G. Roberts, A.J. Coffey, M. Bobrow, D.R. Bentley, Exon structure of the human dystrophin gene Genomics 16 (1993) 536–538, M. Koenig, L.M. Kunkel, Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility, J. Biol. Chem. 265 (1990) 4560–4566]. This would produce fractional STR modules, however, the concept of STRs as proteins domains makes the viability of such fractional motifs questionable. However, certain of these events produce pairs of potentially complementary fractional domain that might reassemble into a hybrid STR motif. We have constructed model fragment corresponding to one such exon-skipping event, and show that the hybrid STR so produced is viable, and furthermore that some of the properties of the protein containing it differ substantially of the native, un-skipped parent.
Keywords: Dystrophin; Spectrin-type-repeat; Domain; Hybrid-domain; Exon skipping; Hinge
Hybrid spectrin type repeats produced by exon-skipping in dystrophin by Nick Menhart (pp. 993-999).
Dystrophin is the protein whose defect underlies Duchenne Muscular Dystrophy, DMD, a common (1:3500 male births) and fatal condition in which muscle tissue deteriorates leading to death in the second or third decade of life. Dystrophin is coded for by the largest human gene, and one of the most complex. It is translated from at least 7 distinct promoters, with the largest transcripts (which are the ones involved in DMD) containing 79 exons over >2.5 Mbp [K.F. O'Brien, L.M. Kunkel, Dystrophin and muscular dystrophy: past, present, and future, Mol. Genet. Metab. 74 (2001) 75–88, H.M. Sadoulet-Puccio, L.M. Kunkel, Dystrophin and its isoforms, Brain Pathol. 6 (1996) 25–35]. Exacerbating this complexity, it has recently been shown that dystrophin is subject to extensive alternative RNA processing, potentially producing a wide variety dystrophin variants [M. Sironi, R. Cagliani, U. Pozzoli, A. Bardoni, G.P. Comi, R. Giorda, N. Bresolin, The dystrophin gene is alternatively spliced throughout its coding sequence FEBS Lett 517 (2002) 163–166]. The structure of the dystrophin protein is highly modular, with the most common module being a motif termed the spectrin type repeat, or STR, of which there are 24. Each STR is roughly coded for by two exons, and the most common type of multiple exon-skipping events start and end at introns in the middle of STRs [R.G. Roberts, A.J. Coffey, M. Bobrow, D.R. Bentley, Exon structure of the human dystrophin gene Genomics 16 (1993) 536–538, M. Koenig, L.M. Kunkel, Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility, J. Biol. Chem. 265 (1990) 4560–4566]. This would produce fractional STR modules, however, the concept of STRs as proteins domains makes the viability of such fractional motifs questionable. However, certain of these events produce pairs of potentially complementary fractional domain that might reassemble into a hybrid STR motif. We have constructed model fragment corresponding to one such exon-skipping event, and show that the hybrid STR so produced is viable, and furthermore that some of the properties of the protein containing it differ substantially of the native, un-skipped parent.
Keywords: Dystrophin; Spectrin-type-repeat; Domain; Hybrid-domain; Exon skipping; Hinge
The effect of the presence of globular proteins and elongated polymers on enzyme activity by Barry K. Derham; John J. Harding (pp. 1000-1006).
We have studied the effect of a crowded (macromolecular) solution on reaction rates of the decarboxylating enzymes urease, pyruvate decarboxylase and glutamate decarboxylase. A variety of crowding agents were used including haemoglobin, lysozyme, various dextrans and polyethylene glycol. Enzyme reaction rates of all three enzymes show two different types of effect that separate the globular proteins from the polysaccharides/polymers. Increasing concentration of globular proteins caused a dramatic rise in enzyme activity up to 30% crowding concentration then the activity decreased, whereas the polymers caused a concentration dependent decrease in activity. The viscosities of the globular proteins were low compared to the polymers. The increased activity with proteins may be due to the decreased amount of free water, which leads to higher effective concentration of substrates, or to an increased oligomeric state by self-association. The lower activities of all enzymes with all agents at high concentrations may be explained by a decrease in rates of diffusion. An increase in protein crowding (decrease in cell water content) may contribute to pathological conditions including cataract and Alzheimer's disease.
Keywords: Abbreviations; PEG; polyethlyene glycol; MES; 2-Morpholinoethanesulfonic acidEnzyme; Decarboxylase; Globular; Protein; Polymer; Crowding
The effect of the presence of globular proteins and elongated polymers on enzyme activity by Barry K. Derham; John J. Harding (pp. 1000-1006).
We have studied the effect of a crowded (macromolecular) solution on reaction rates of the decarboxylating enzymes urease, pyruvate decarboxylase and glutamate decarboxylase. A variety of crowding agents were used including haemoglobin, lysozyme, various dextrans and polyethylene glycol. Enzyme reaction rates of all three enzymes show two different types of effect that separate the globular proteins from the polysaccharides/polymers. Increasing concentration of globular proteins caused a dramatic rise in enzyme activity up to 30% crowding concentration then the activity decreased, whereas the polymers caused a concentration dependent decrease in activity. The viscosities of the globular proteins were low compared to the polymers. The increased activity with proteins may be due to the decreased amount of free water, which leads to higher effective concentration of substrates, or to an increased oligomeric state by self-association. The lower activities of all enzymes with all agents at high concentrations may be explained by a decrease in rates of diffusion. An increase in protein crowding (decrease in cell water content) may contribute to pathological conditions including cataract and Alzheimer's disease.
Keywords: Abbreviations; PEG; polyethlyene glycol; MES; 2-Morpholinoethanesulfonic acidEnzyme; Decarboxylase; Globular; Protein; Polymer; Crowding
Constitutive and inducible stress proteins dominate the proteome of the murine inner medullary collecting duct-3 (mIMCD3) cell line by Nelly Valkova; Dietmar Kültz (pp. 1007-1020).
A proteome map of the most abundant proteins in the murine inner medullary collecting duct (mIMCD3) cell line was generated by 2-dimensional gel electrophoresis (2D-GE) combined with MALDI-TOF/TOF mass spectrometry. The 2-D model map identifies 77 distinct constitutive proteins and a total of 86 spots including isoforms. Protein identification was based on both peptide mass fingerprinting (MS) and peptide fragmentation (MS/MS) data. High confidence Mascot scores were obtained in the database search, due to the high quality and the number of MS/MS spectra which provided matching sequence information to the database. A functional classification of the identified proteins showed that a high proportion were stress proteins, such as heat shock proteins and proteins with anti-oxidant activity. Other proteins identified were involved in cytoskeletal maintenance, metabolism and energy generation, as well as in translation, transcription, RNA processing and other cell cycle processes. Exposure of the mIMCD3 cells to hyperosmotic stress using 600 mOsmol/kg NaCl or Urea or 700 mOsmol/kg NaCl–Urea (50:50) resulted in the greatest proteome upregulation in 700 mosM NaCl–Urea and the greatest downregulation in 600 mosM NaCl. Several proteins with molecular chaperone function were induced, such as alpha-B crystallin, two Hsp70 isoforms, the osmotic stress protein (Osp94), as well as aldose reductase. Additional isoforms of the translation elongation factors Eef2 and Eef1a1 were induced. Characterization of the phosphoproteome of mIMCD3 cells with a phosphoprotein-specific stain showed a significant proportion of the proteome was phosphorylated. Additionally, exposure of mIMCD3 cells to 600 mOsmol/kg NaCl hyperosmotic stress resulted in a 1.8-fold higher phosphorylation level of the most acidic isoform of the heat shock protein Hsp27 compared to its phosphorylation level under iso-osmotic conditions.
Keywords: 2-D gel electrophoresis; Proteome; Inner medulla; Collecting duct; Hyperosmotic stress; Mass spectrometry; Phosphoprotein
Constitutive and inducible stress proteins dominate the proteome of the murine inner medullary collecting duct-3 (mIMCD3) cell line by Nelly Valkova; Dietmar Kültz (pp. 1007-1020).
A proteome map of the most abundant proteins in the murine inner medullary collecting duct (mIMCD3) cell line was generated by 2-dimensional gel electrophoresis (2D-GE) combined with MALDI-TOF/TOF mass spectrometry. The 2-D model map identifies 77 distinct constitutive proteins and a total of 86 spots including isoforms. Protein identification was based on both peptide mass fingerprinting (MS) and peptide fragmentation (MS/MS) data. High confidence Mascot scores were obtained in the database search, due to the high quality and the number of MS/MS spectra which provided matching sequence information to the database. A functional classification of the identified proteins showed that a high proportion were stress proteins, such as heat shock proteins and proteins with anti-oxidant activity. Other proteins identified were involved in cytoskeletal maintenance, metabolism and energy generation, as well as in translation, transcription, RNA processing and other cell cycle processes. Exposure of the mIMCD3 cells to hyperosmotic stress using 600 mOsmol/kg NaCl or Urea or 700 mOsmol/kg NaCl–Urea (50:50) resulted in the greatest proteome upregulation in 700 mosM NaCl–Urea and the greatest downregulation in 600 mosM NaCl. Several proteins with molecular chaperone function were induced, such as alpha-B crystallin, two Hsp70 isoforms, the osmotic stress protein (Osp94), as well as aldose reductase. Additional isoforms of the translation elongation factors Eef2 and Eef1a1 were induced. Characterization of the phosphoproteome of mIMCD3 cells with a phosphoprotein-specific stain showed a significant proportion of the proteome was phosphorylated. Additionally, exposure of mIMCD3 cells to 600 mOsmol/kg NaCl hyperosmotic stress resulted in a 1.8-fold higher phosphorylation level of the most acidic isoform of the heat shock protein Hsp27 compared to its phosphorylation level under iso-osmotic conditions.
Keywords: 2-D gel electrophoresis; Proteome; Inner medulla; Collecting duct; Hyperosmotic stress; Mass spectrometry; Phosphoprotein
Kinetic stability plays a dominant role in the denaturant-induced unfolding of Erythrina indica lectin by Sujit Ghosh; Dipak K. Mandal (pp. 1021-1028).
The urea-induced denaturation of dimeric Erythrina indica lectin (EIL) has been studied at pH 7.2 under equilibrium and kinetic conditions in the temperature range of 40–55 °C. The structure of EIL is largely unaffected in this temperature range in absence of denaturant, and also in 8 M urea after incubation for 24 h at ambient temperature. The equilibrium denaturation of EIL exhibits a monophasic unfolding transition from the native dimer to the unfolded monomer as monitored by fluorescence, far-UV CD, and size-exclusion FPLC. The thermodynamic parameters determined for the two-state unfolding equilibrium show that the free energy of unfolding (Δ Gu, aq) remains practically same between 40 and 55 °C, with a value of 11.8 ± 0.6 kcal mol−1 (monomer units). The unfolding kinetics of EIL describes a single exponential decay pattern, and the apparent rate constants determined at different temperatures indicate that the rate of the unfolding reaction increases several fold with increase in temperature. The presence of probe like external metal ions (Mn2+, Ca2+) does not influence the unfolding reaction thermodynamically or kinetically; however, the presence of EDTA affects only kinetics. The present results suggest that the ability of EIL to preserve the structural integrity against the highly denaturing conditions is linked primarily to its kinetic stability, and the synergic action of heat and denaturant is involved in the unfolding of the protein.
Keywords: Abbreviations; EIL; Erythrina indica; lectin; HEPES; N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; EDTA; ethylenediaminetetraacetic acid (disodium salt); CD; circular dichroism; FPLC; fast-protein liquid chromatographyErythrina indica lectin; Legume lectin; Equilibrium denaturation; Unfolding kinetics; Thermodynamic and kinetic stability
Kinetic stability plays a dominant role in the denaturant-induced unfolding of Erythrina indica lectin by Sujit Ghosh; Dipak K. Mandal (pp. 1021-1028).
The urea-induced denaturation of dimeric Erythrina indica lectin (EIL) has been studied at pH 7.2 under equilibrium and kinetic conditions in the temperature range of 40–55 °C. The structure of EIL is largely unaffected in this temperature range in absence of denaturant, and also in 8 M urea after incubation for 24 h at ambient temperature. The equilibrium denaturation of EIL exhibits a monophasic unfolding transition from the native dimer to the unfolded monomer as monitored by fluorescence, far-UV CD, and size-exclusion FPLC. The thermodynamic parameters determined for the two-state unfolding equilibrium show that the free energy of unfolding (Δ Gu, aq) remains practically same between 40 and 55 °C, with a value of 11.8 ± 0.6 kcal mol−1 (monomer units). The unfolding kinetics of EIL describes a single exponential decay pattern, and the apparent rate constants determined at different temperatures indicate that the rate of the unfolding reaction increases several fold with increase in temperature. The presence of probe like external metal ions (Mn2+, Ca2+) does not influence the unfolding reaction thermodynamically or kinetically; however, the presence of EDTA affects only kinetics. The present results suggest that the ability of EIL to preserve the structural integrity against the highly denaturing conditions is linked primarily to its kinetic stability, and the synergic action of heat and denaturant is involved in the unfolding of the protein.
Keywords: Abbreviations; EIL; Erythrina indica; lectin; HEPES; N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; EDTA; ethylenediaminetetraacetic acid (disodium salt); CD; circular dichroism; FPLC; fast-protein liquid chromatographyErythrina indica lectin; Legume lectin; Equilibrium denaturation; Unfolding kinetics; Thermodynamic and kinetic stability
Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles by Nan Chen; Jun-Bao Fan; Jin Xiang; Jie Chen; Yi Liang (pp. 1029-1035).
The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent mediae, respectively. The influences of the molar ratio of water to surfactant ω0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show “superactivity? in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of ω0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at ω0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.
Keywords: Abbreviations; AOT; Aerosol OT or sodium bis(2-ethylhexyl)sulfosuccinateCellulase; Microcrystalline cellulose; Reverse micelle; Enzymatic hydrolysis
Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles by Nan Chen; Jun-Bao Fan; Jin Xiang; Jie Chen; Yi Liang (pp. 1029-1035).
The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent mediae, respectively. The influences of the molar ratio of water to surfactant ω0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show “superactivity” in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of ω0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at ω0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.
Keywords: Abbreviations; AOT; Aerosol OT or sodium bis(2-ethylhexyl)sulfosuccinateCellulase; Microcrystalline cellulose; Reverse micelle; Enzymatic hydrolysis
Functional characterization of the glycosyltransferase domain of penicillin-binding protein 1a from Thermotoga maritima by Julien Offant; François Michoux; Annabelle Dermiaux; Jacques Biton; Yves Bourne (pp. 1036-1042).
Class A penicillin-binding proteins (A-PBPs) are high-molecular weight membrane-bound bifunctional enzymes that catalyze the penicillin-sensitive transpeptidation and transglycosylation reaction steps involved in peptidoglycan assembling. We have over-expressed and characterized a soluble form of the glycosyltransferase domain of PBP1a (GT-PBP1a*) from the hyperthermophilic bacteria Thermotoga maritima. GT-PBP1a* efficiently catalyses peptidoglycan biosynthesis, as shown using an in vitro biosynthetized dansylated-lipid II substrate and a HPLC-coupled assay, and is specifically inhibited by moenomycin. GT-PBP1a* tends to spontaneously aggregate in detergent-free solution, a feature that supports existence of a secondary site for membrane association, distinct from the N-terminal transmembrane anchoring region. Overall, our preliminary data document the biochemical properties of GT-PBP1a* and should guide further studies aimed at deciphering the structural determinants involved into membrane binding by this class of enzymes.
Keywords: Abbreviations; PBP; penicillin-binding protein; MGT; monofunctional glycosyltransferase; GTase; glycosyltransferase; HCA; hydrophobic cluster analysis; SPA; scintillation proximity assay; DLS; dynamic light scatteringPenicillin binding protein; Glycosyltransferase; Antibiotic; Peptidoglycan biosynthesis; Oligomerization; Detergent
Functional characterization of the glycosyltransferase domain of penicillin-binding protein 1a from Thermotoga maritima by Julien Offant; François Michoux; Annabelle Dermiaux; Jacques Biton; Yves Bourne (pp. 1036-1042).
Class A penicillin-binding proteins (A-PBPs) are high-molecular weight membrane-bound bifunctional enzymes that catalyze the penicillin-sensitive transpeptidation and transglycosylation reaction steps involved in peptidoglycan assembling. We have over-expressed and characterized a soluble form of the glycosyltransferase domain of PBP1a (GT-PBP1a*) from the hyperthermophilic bacteria Thermotoga maritima. GT-PBP1a* efficiently catalyses peptidoglycan biosynthesis, as shown using an in vitro biosynthetized dansylated-lipid II substrate and a HPLC-coupled assay, and is specifically inhibited by moenomycin. GT-PBP1a* tends to spontaneously aggregate in detergent-free solution, a feature that supports existence of a secondary site for membrane association, distinct from the N-terminal transmembrane anchoring region. Overall, our preliminary data document the biochemical properties of GT-PBP1a* and should guide further studies aimed at deciphering the structural determinants involved into membrane binding by this class of enzymes.
Keywords: Abbreviations; PBP; penicillin-binding protein; MGT; monofunctional glycosyltransferase; GTase; glycosyltransferase; HCA; hydrophobic cluster analysis; SPA; scintillation proximity assay; DLS; dynamic light scatteringPenicillin binding protein; Glycosyltransferase; Antibiotic; Peptidoglycan biosynthesis; Oligomerization; Detergent
In vitro characterization of the cysteine-rich capping domains in a plant leucine rich repeat protein by Olatomirin O. Kolade; Vicki A. Bamford; Gema Ancillo Anton; Jonathan D.G. Jones; Pablo Vera; Andrew M. Hemmings (pp. 1043-1053).
Plant leucine rich repeat (LRR) proteins have diverse functions and cellular locations. An important unresolved question involves the role of the cysteine-rich capping domains which flank the LRR domain. Such studies have been hampered by difficulties in producing recombinant LRR proteins in yields sufficient for biochemical analysis. We have used Escherichia coli to overproduce Leucine Rich Protein (LRP), a small model LRR protein from tomato containing approximately five LRRs. The LRP capping domain sequences resemble those from plant disease resistance proteins and receptor-like protein kinases. LRP was purified as a soluble, crystallizable, monomeric protein by renaturation of a GST-fusion protein. The four cysteine residues in LRP were found to form two disulfide bonds, one each in the N- and C-terminal LRR-capping domains, the presence of which is necessary to protect the LRR domain from proteolysis in vitro. Fluorescence and CD spectroscopies together with molecular modelling revealed that structural features of the N-capping domain may be destabilised on reduction. These include a tryptophan stacking interaction and a long α-helix of residues 30–44. LRP deletion mutants lacking the capping domains showed a propensity to aggregate and increased proteolytic sensitivity. These results have important implications for future structure–function studies of plant LRR proteins.
Keywords: Tomato Leucine Rich Protein; leucine rich repeat; LRR; Capping domains; Disulfide bridge
In vitro characterization of the cysteine-rich capping domains in a plant leucine rich repeat protein by Olatomirin O. Kolade; Vicki A. Bamford; Gema Ancillo Anton; Jonathan D.G. Jones; Pablo Vera; Andrew M. Hemmings (pp. 1043-1053).
Plant leucine rich repeat (LRR) proteins have diverse functions and cellular locations. An important unresolved question involves the role of the cysteine-rich capping domains which flank the LRR domain. Such studies have been hampered by difficulties in producing recombinant LRR proteins in yields sufficient for biochemical analysis. We have used Escherichia coli to overproduce Leucine Rich Protein (LRP), a small model LRR protein from tomato containing approximately five LRRs. The LRP capping domain sequences resemble those from plant disease resistance proteins and receptor-like protein kinases. LRP was purified as a soluble, crystallizable, monomeric protein by renaturation of a GST-fusion protein. The four cysteine residues in LRP were found to form two disulfide bonds, one each in the N- and C-terminal LRR-capping domains, the presence of which is necessary to protect the LRR domain from proteolysis in vitro. Fluorescence and CD spectroscopies together with molecular modelling revealed that structural features of the N-capping domain may be destabilised on reduction. These include a tryptophan stacking interaction and a long α-helix of residues 30–44. LRP deletion mutants lacking the capping domains showed a propensity to aggregate and increased proteolytic sensitivity. These results have important implications for future structure–function studies of plant LRR proteins.
Keywords: Tomato Leucine Rich Protein; leucine rich repeat; LRR; Capping domains; Disulfide bridge
Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle by Yurii S. Borovikov; Natalia Kulikova; Olga E. Pronina; Svetlana S. Khaimina; Antoni Wrzosek; Renata Dabrowska (pp. 1054-1062).
Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7° relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**·M**·ADP·Pi (weak binding) and A∧·M∧·ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the “closed? position caldesmon “freezes? the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.
Keywords: Abbreviations; S1; myosin subfragment 1; LC; 20; 20,000-dalton light chain of smooth muscle myosin; FITC; fluorescein-5-isothiocyanate; IAEDANS; N-iodoacetyl-; N; ′-(5-sulfo-1-naphtylo)ethylenediamine; NEM; N; -ethylmaleimide; pPDM; N; ,; N; ′-1,4-phenylenedimaleimide; DTT; dithiothreitolCaldesmon; F-actin; Myosin subfragment S1; Conformational change; Ghost muscle fiber; Fluorescence polarization
Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle by Yurii S. Borovikov; Natalia Kulikova; Olga E. Pronina; Svetlana S. Khaimina; Antoni Wrzosek; Renata Dabrowska (pp. 1054-1062).
Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7° relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**·M**·ADP·Pi (weak binding) and A∧·M∧·ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the “closed” position caldesmon “freezes” the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.
Keywords: Abbreviations; S1; myosin subfragment 1; LC; 20; 20,000-dalton light chain of smooth muscle myosin; FITC; fluorescein-5-isothiocyanate; IAEDANS; N-iodoacetyl-; N; ′-(5-sulfo-1-naphtylo)ethylenediamine; NEM; N; -ethylmaleimide; pPDM; N; ,; N; ′-1,4-phenylenedimaleimide; DTT; dithiothreitolCaldesmon; F-actin; Myosin subfragment S1; Conformational change; Ghost muscle fiber; Fluorescence polarization
The papaya Kunitz-type trypsin inhibitor is a highly stable β-sheet glycoprotein by Mohamed Azarkan; Rachid Dibiani; Erik Goormaghtigh; Vincent Raussens; Danielle Baeyens-Volant (pp. 1063-1072).
The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive β-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5–11.0) and temperature (up to 80 °C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.
Keywords: Carica papaya; Latex; Kunitz-type inhibitor; All-β conformation; Circular dichroism; Infrared spectroscopy
The papaya Kunitz-type trypsin inhibitor is a highly stable β-sheet glycoprotein by Mohamed Azarkan; Rachid Dibiani; Erik Goormaghtigh; Vincent Raussens; Danielle Baeyens-Volant (pp. 1063-1072).
The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive β-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5–11.0) and temperature (up to 80 °C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.
Keywords: Carica papaya; Latex; Kunitz-type inhibitor; All-β conformation; Circular dichroism; Infrared spectroscopy
The E3 ubiquitin ligase CHIP binds the androgen receptor in a phosphorylation-dependent manner by Ian Rees; Sukyeong Lee; Hyeung Kim; Francis T.F. Tsai (pp. 1073-1079).
In Eukarya, the 26S proteasome is primarily responsible for intracellular protein degradation. To be degraded, proteins must be ubiquitinated. The latter requires a multi-enzyme cascade consisting of an E1, an E2, and an E3 enzyme. While there is only a single E1 and a few E2s, there are many different E3s that target substrates by recognizing specific sequence motifs, known as degrons. Here, we have used the peptide array technology to identify binding motifs in the human androgen receptor (AR), which are recognized by the Carboxyl-terminus of Hsc70-Interacting Protein (CHIP), a U-box E3 and Hsp70/Hsp90 co-chaperone. We show that CHIP recognizes AR in a highly specific, phosphorylation- and sequence-dependent manner, and propose that this interaction could provide a mechanism that regulates the degradation of CHIP substrates.
Keywords: CHIP; Androgen receptor; Hsp90 co-chaperone; E3 ubiquitin ligase; Peptide array
The E3 ubiquitin ligase CHIP binds the androgen receptor in a phosphorylation-dependent manner by Ian Rees; Sukyeong Lee; Hyeung Kim; Francis T.F. Tsai (pp. 1073-1079).
In Eukarya, the 26S proteasome is primarily responsible for intracellular protein degradation. To be degraded, proteins must be ubiquitinated. The latter requires a multi-enzyme cascade consisting of an E1, an E2, and an E3 enzyme. While there is only a single E1 and a few E2s, there are many different E3s that target substrates by recognizing specific sequence motifs, known as degrons. Here, we have used the peptide array technology to identify binding motifs in the human androgen receptor (AR), which are recognized by the Carboxyl-terminus of Hsc70-Interacting Protein (CHIP), a U-box E3 and Hsp70/Hsp90 co-chaperone. We show that CHIP recognizes AR in a highly specific, phosphorylation- and sequence-dependent manner, and propose that this interaction could provide a mechanism that regulates the degradation of CHIP substrates.
Keywords: CHIP; Androgen receptor; Hsp90 co-chaperone; E3 ubiquitin ligase; Peptide array
A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency by Kalyani Mondal; Payal Mehta; Bodh Raj Mehta; Deepak Varandani; Munishwar Nath Gupta (pp. 1080-1086).
A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/ Km value ( Vmax/ Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 °C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.
Keywords: Pseudomonas cepacia; lipase; Alginate; Noncovalent immobilization; Atomic force microscopy; Smart biocatalysts; Lipase bioconjugate
A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency by Kalyani Mondal; Payal Mehta; Bodh Raj Mehta; Deepak Varandani; Munishwar Nath Gupta (pp. 1080-1086).
A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/ Km value ( Vmax/ Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 °C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.
Keywords: Pseudomonas cepacia; lipase; Alginate; Noncovalent immobilization; Atomic force microscopy; Smart biocatalysts; Lipase bioconjugate
The first archaeall-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: Gene cloning and enzymological characterization by Kazunari Yoneda; Ryushi Kawakami; Yuya Tagashira; Haruhiko Sakuraba; Shuichiro Goda; Toshihisa Ohshima (pp. 1087-1093).
A gene encoding anl-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostablel-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 °C. The optimum temperature forl-aspartate dehydrogenation was about 80 °C. The enzyme specifically utilizedl-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values forl-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro- R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeall-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.
Keywords: Abbreviations; l; -aspDH; l; -aspartate dehydrogenase; l; -gluDH; l; -glutamate dehydrogenase; GOT; glutamate oxaloacetate aminotransferase; LAO; l; -aspartate oxidase; QS; quinolinate synthasel; -Aspartate dehydrogenase; Archaeoglobus fulgidus; NAD biosynthesis; Archaea; Hyperthermophile; Amino acid dehydrogenase
The first archaeall-aspartate dehydrogenase from the hyperthermophile Archaeoglobus fulgidus: Gene cloning and enzymological characterization by Kazunari Yoneda; Ryushi Kawakami; Yuya Tagashira; Haruhiko Sakuraba; Shuichiro Goda; Toshihisa Ohshima (pp. 1087-1093).
A gene encoding anl-aspartate dehydrogenase (EC 1.4.1.21) homologue was identified in the anaerobic hyperthermophilic archaeon Archaeoglobus fulgidus. After expression in Escherichia coli, the gene product was purified to homogeneity, yielding a homodimeric protein with a molecular mass of about 48 kDa. Characterization revealed the enzyme to be a highly thermostablel-aspartate dehydrogenase, showing little loss of activity following incubation for 1 h at up to 80 °C. The optimum temperature forl-aspartate dehydrogenation was about 80 °C. The enzyme specifically utilizedl-aspartate as the electron donor, while either NAD or NADP could serve as the electron acceptor. The Km values forl-aspartate were 0.19 and 4.3 mM when NAD or NADP, respectively, served as the electron acceptor. The Km values for NAD and NADP were 0.11 and 0.32 mM, respectively. For reductive amination, the Km values for oxaloacetate, NADH and ammonia were 1.2, 0.014 and 167 mM, respectively. The enzyme showed pro- R (A-type) stereospecificity for hydrogen transfer from the C4 position of the nicotinamide moiety of NADH. This is the first report of an archaeall-aspartate dehydrogenase. Within the archaeal domain, homologues of this enzyme occurred in many Methanogenic species, but not in Thermococcales or Sulfolobales species.
Keywords: Abbreviations; l; -aspDH; l; -aspartate dehydrogenase; l; -gluDH; l; -glutamate dehydrogenase; GOT; glutamate oxaloacetate aminotransferase; LAO; l; -aspartate oxidase; QS; quinolinate synthasel; -Aspartate dehydrogenase; Archaeoglobus fulgidus; NAD biosynthesis; Archaea; Hyperthermophile; Amino acid dehydrogenase
pH-dependent association of factor VIII chains: Enhancement of affinity at physiological pH by Cu2+ by Hironao Wakabayashi; Qian Zhou; Keiji Nogami; Charles Ansong; Fatbardha Varfaj; Stephen Miles; Philip J. Fay (pp. 1094-1101).
Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5–6.75 ( Kd ∼4 nM), whereas a Kd ∼20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 μM total Cu2+) on maximal activity regenerated was negligible at pH 6.25–8.0. However, this level of Cu2+ increased the inter-chain affinity by ∼5-fold at pH 7.25. This effect resulted from an ∼1.5-fold increased association rate constant ( kon) and an ∼3-fold reduced dissociation rate constant ( koff). High affinity ( Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.
Keywords: Abbreviations; HC; factor VIII heavy chain; LC; factor VIII light chain; EDTA; ethylenediamine tetraacetic acid; MES; 2-[N-morpholino]ethanesulfonic acid; MOPS; 3-[N-morpholino]propanesulfonic acid; HEPES; N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; BSA; bovine serum albumin; PS; phosphatidylserine; PC; phosphatidylcholine; PE; phosphatidylethanolamineFactor VIII; Copper; Factor Xa generation; Binding affinity; Binding kinetics; pH
pH-dependent association of factor VIII chains: Enhancement of affinity at physiological pH by Cu2+ by Hironao Wakabayashi; Qian Zhou; Keiji Nogami; Charles Ansong; Fatbardha Varfaj; Stephen Miles; Philip J. Fay (pp. 1094-1101).
Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5–6.75 ( Kd ∼4 nM), whereas a Kd ∼20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 μM total Cu2+) on maximal activity regenerated was negligible at pH 6.25–8.0. However, this level of Cu2+ increased the inter-chain affinity by ∼5-fold at pH 7.25. This effect resulted from an ∼1.5-fold increased association rate constant ( kon) and an ∼3-fold reduced dissociation rate constant ( koff). High affinity ( Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.
Keywords: Abbreviations; HC; factor VIII heavy chain; LC; factor VIII light chain; EDTA; ethylenediamine tetraacetic acid; MES; 2-[N-morpholino]ethanesulfonic acid; MOPS; 3-[N-morpholino]propanesulfonic acid; HEPES; N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; BSA; bovine serum albumin; PS; phosphatidylserine; PC; phosphatidylcholine; PE; phosphatidylethanolamineFactor VIII; Copper; Factor Xa generation; Binding affinity; Binding kinetics; pH
Allosteric communication between alpha and beta subunits of tryptophan synthase: Modelling the open-closed transition of the alpha subunit by Francesca Spyrakis; Samanta Raboni; Pietro Cozzini; Stefano Bettati; Andrea Mozzarelli (pp. 1102-1109).
Ligand binding to the α-subunit of the α2β2 complex of tryptophan synthase induces the αloop6 closure over the α-active site. This conformational change is associated with the formation of a hydrogen bond between αGly181 NH group and βSer178 carbonyl oxygen, a key event for the triggering of intersubunit allosteric signals. Mutation of βSer178 to Pro and αGly181 to Pro, Ala, Phe and Val abolishes the ligand-induced intersubunit communication. Molecular dynamics methods were applied to simulate the conformation of the highly flexible and crystallographically undetectable open state of αloop6 in the wild type and in the α181 mutants. The open conformation of αloop6 is favoured in the wild type enzyme in the absence of α-ligands, and in the α181 mutants both in the presence and absence of bound ligands. A very good correlation was found between the extent of limited tryptic proteolysis and both the hydrogen bond distance between αX181 and βSer178, obtained from the molecular dynamics simulation, and the hydrogen bond strength, evaluated by HINT, an empirical force field that takes into account both enthalpic and entropic contributions. Comparison of the open and closed conformations of αloop6 suggests a pathway for substrate entrance into the α-active site and provides an explanation for the limited catalytic efficiency of the open state.
Keywords: Abbreviations; TRPS; tryptophan synthase; IAD; indole acetylaspartate; MD; molecular dynamicsPyridoxal 5′-phosphate; Open-closed transition; Allosteric communication; Dynamic simulation; HINT
Allosteric communication between alpha and beta subunits of tryptophan synthase: Modelling the open-closed transition of the alpha subunit by Francesca Spyrakis; Samanta Raboni; Pietro Cozzini; Stefano Bettati; Andrea Mozzarelli (pp. 1102-1109).
Ligand binding to the α-subunit of the α2β2 complex of tryptophan synthase induces the αloop6 closure over the α-active site. This conformational change is associated with the formation of a hydrogen bond between αGly181 NH group and βSer178 carbonyl oxygen, a key event for the triggering of intersubunit allosteric signals. Mutation of βSer178 to Pro and αGly181 to Pro, Ala, Phe and Val abolishes the ligand-induced intersubunit communication. Molecular dynamics methods were applied to simulate the conformation of the highly flexible and crystallographically undetectable open state of αloop6 in the wild type and in the α181 mutants. The open conformation of αloop6 is favoured in the wild type enzyme in the absence of α-ligands, and in the α181 mutants both in the presence and absence of bound ligands. A very good correlation was found between the extent of limited tryptic proteolysis and both the hydrogen bond distance between αX181 and βSer178, obtained from the molecular dynamics simulation, and the hydrogen bond strength, evaluated by HINT, an empirical force field that takes into account both enthalpic and entropic contributions. Comparison of the open and closed conformations of αloop6 suggests a pathway for substrate entrance into the α-active site and provides an explanation for the limited catalytic efficiency of the open state.
Keywords: Abbreviations; TRPS; tryptophan synthase; IAD; indole acetylaspartate; MD; molecular dynamicsPyridoxal 5′-phosphate; Open-closed transition; Allosteric communication; Dynamic simulation; HINT
Effect of temperature and pH on the secondary structure and processes of oligomerization of 19 kDa alpha-zein by Vanessa Cabra; Roberto Arreguin; Rafael Vazquez-Duhalt; Amelia Farres (pp. 1110-1118).
Highly hydrophobic protein Z19 zein shows a tendency towards oligomerization. The role of temperature and pH on the oligomerization process was studied monitoring the secondary structure content and the appearance of aggregates by Circular Dichroism Spectroscopy (CD) and Dinamic Light Scattering (DLS). Z19 zein suffers irreversible thermal denaturalization, as demonstrated by far-UV CD measurements. DLS data indicate that this denaturalization is accompanied by oligomerization processes which are strongly dependent on temperature. The aggregates that appear when the sample is heated maintain a certain amount of their native structure. Oligomers, showing high stability to temperature changes and other denaturing conditions with molecular weights of 45, 66 kDa and higher, were detected by SDS-PAGE. The secondary structure strongly depends on pH. Thus, at pH above pI (6.8), all the protein structure is in alpha helix. The formation of disulfide bonds plays an important role in the aggregation process, since most of the sulfhydryls in the protein (97.52%) form disulfide bonds and only 2.47% of them are free and superficially exposed. The sensitivity towards thermal denaturalization is also affected by pH rises.
Keywords: Abbreviations; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD; circular dichroism; BME; beta mercapthoethanol; HMW; High Molecular Weight; DLS; Dinamic Light Scattering; EDTA; Ethilendiaminotethracetic acid; DTNB; 5′5′-dithiobis(2-nitrobenzoic acid)Alpha-zeins; Protein aggregation; Circular Dichroism; Dinamic light scattering; Disulfide bond
Effect of temperature and pH on the secondary structure and processes of oligomerization of 19 kDa alpha-zein by Vanessa Cabra; Roberto Arreguin; Rafael Vazquez-Duhalt; Amelia Farres (pp. 1110-1118).
Highly hydrophobic protein Z19 zein shows a tendency towards oligomerization. The role of temperature and pH on the oligomerization process was studied monitoring the secondary structure content and the appearance of aggregates by Circular Dichroism Spectroscopy (CD) and Dinamic Light Scattering (DLS). Z19 zein suffers irreversible thermal denaturalization, as demonstrated by far-UV CD measurements. DLS data indicate that this denaturalization is accompanied by oligomerization processes which are strongly dependent on temperature. The aggregates that appear when the sample is heated maintain a certain amount of their native structure. Oligomers, showing high stability to temperature changes and other denaturing conditions with molecular weights of 45, 66 kDa and higher, were detected by SDS-PAGE. The secondary structure strongly depends on pH. Thus, at pH above pI (6.8), all the protein structure is in alpha helix. The formation of disulfide bonds plays an important role in the aggregation process, since most of the sulfhydryls in the protein (97.52%) form disulfide bonds and only 2.47% of them are free and superficially exposed. The sensitivity towards thermal denaturalization is also affected by pH rises.
Keywords: Abbreviations; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD; circular dichroism; BME; beta mercapthoethanol; HMW; High Molecular Weight; DLS; Dinamic Light Scattering; EDTA; Ethilendiaminotethracetic acid; DTNB; 5′5′-dithiobis(2-nitrobenzoic acid)Alpha-zeins; Protein aggregation; Circular Dichroism; Dinamic light scattering; Disulfide bond
Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles by Ashok Ganesan; Nicholas C. Price; Sharon M. Kelly; Inga Petry; Barry D. Moore; Peter J. Halling (pp. 1119-1125).
Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 μm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.
Keywords: Immobilised enzyme; Circular dichroism; Subtilisin Carlsberg; Silica gel; Differential scattering; Absorption flattening; Secondary structure; Organic media
Circular dichroism studies of subtilisin Carlsberg immobilised on micron sized silica particles by Ashok Ganesan; Nicholas C. Price; Sharon M. Kelly; Inga Petry; Barry D. Moore; Peter J. Halling (pp. 1119-1125).
Immobilised enzymes are widely used in industry, but the reasons for loss of activity of such biocatalysts are usually not known. We have used circular dichroism (CD) to investigate the structure of one such system, i.e., subtilisin Carlsberg (SC) immobilised on silica gel particles (60 μm). A number of technical problems have to be overcome in order to obtain appropriate data from which conclusions can be drawn. A rotating cell holder has been developed to avoid sedimentation of the silica particles during the collection of spectra. By moving the cell holder as close as possible to the detector window, the effects of differential scattering can be minimised. However, the effects of absorption flattening limit the extent to which reliable quantitative information on secondary structure content can be obtained from far UV CD studies. We have used an empirical approach based on absorbance units derived from the high-tension voltage to correct for absorption flattening effects. After applying the correction there was satisfactory agreement with the solution spectra. Comparison of the fresh and used (inactive) SC-silica gel spectra in organic media reveals substantial change in the secondary structure. Additional evidence for loss of native conformation is provided by the significant decrease in the near UV CD spectrum. These results for the first time clearly demonstrate the origin of enzyme instability in the immobilised state.
Keywords: Immobilised enzyme; Circular dichroism; Subtilisin Carlsberg; Silica gel; Differential scattering; Absorption flattening; Secondary structure; Organic media
The interaction of cytochrome P450 17α with NADPH-cytochrome P450 reductase, investigated using chemical modification and MALDI-TOF mass spectrometry by Laleh Nikfarjam; Shunsuke Izumi; Takeshi Yamazaki; Shiro Kominami (pp. 1126-1131).
The lysine residues of guinea pig P450 17α were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17α prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17α was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17α in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17α with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.
Keywords: Cytochrome P450; NADPH-cyrochrome P450 reductase; Chemical modification; MALDI-TOF mass spectrometry; Protein-protein interaction
The interaction of cytochrome P450 17α with NADPH-cytochrome P450 reductase, investigated using chemical modification and MALDI-TOF mass spectrometry by Laleh Nikfarjam; Shunsuke Izumi; Takeshi Yamazaki; Shiro Kominami (pp. 1126-1131).
The lysine residues of guinea pig P450 17α were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17α prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17α was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17α in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17α with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.
Keywords: Cytochrome P450; NADPH-cyrochrome P450 reductase; Chemical modification; MALDI-TOF mass spectrometry; Protein-protein interaction
Mutational analysis and membrane-interactions of the β-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P by Gunnar Fimland; Jussi Pirneskoski; Jasadee Kaewsrichan; Arimatti Jutila; Per Eugen Kristiansen; Paavo K.J. Kinnunen; Jon Nissen-Meyer (pp. 1132-1140).
To gain insight into how the N-terminal three-stranded β-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide–liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the β-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.
Keywords: Abbreviations; AMP; antimicrobial peptide; MIC; minimal inhibitory concentration; NMR; nuclear magnetic resonance; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; rac; -glycerol; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholineBacteriocin; Antimicrobial peptide; Peptide–membrane interaction; Sakacin P; Mutagenesis
Mutational analysis and membrane-interactions of the β-sheet-like N-terminal domain of the pediocin-like antimicrobial peptide sakacin P by Gunnar Fimland; Jussi Pirneskoski; Jasadee Kaewsrichan; Arimatti Jutila; Per Eugen Kristiansen; Paavo K.J. Kinnunen; Jon Nissen-Meyer (pp. 1132-1140).
To gain insight into how the N-terminal three-stranded β-sheet-like domain in pediocin-like antimicrobial peptides positions itself on membranes, residues in the well-conserved (Y)YGNGV-motif in the domain were substituted and the effect of the substitutions on antimicrobial activity and binding of peptides to liposomes was determined. Peptide–liposome interactions were detected by measuring tryptophan-fluorescence upon exposing liposomes to peptides in which a tryptophan residue had been introduced in the N-terminal domain. The results revealed that the N-terminal domain associates readily with anionic liposomes, but not with neutral liposomes. The electrostatic interactions between peptides and liposomes facilitated the penetration of some of the peptide residues into the liposomes. Measuring the antimicrobial activity of the mutated peptides revealed that the Tyr2Leu and Tyr3Leu mutations resulted in about a 10-fold reduction in activity, whereas the Tyr2Trp, Tyr2Phe, Tyr3Trp and Tyr3Phe mutations were tolerated fairly well, especially the mutations in position 3. The Val7Ile mutation did not have a marked detrimental effect on the activity. The Gly6Ala mutation was highly detrimental, consistent with Gly6 being in one of the turns in the β-sheet-like N-terminal domain, whereas the Gly4Ala mutation was tolerated fairly well. All mutations involving Asn5, including the conservative mutations Asn5Gln and Asn5Asp, were very deleterious. Thus, both the polar amide group on the side chain of Asn5 and its exact position in space were crucial for the peptides to be fully active. Taken together, the results are consistent with Val7 positioning itself in the hydrophobic core of target membranes, thus forcing most of the other residues in the N-terminal domain into the membrane interface region: Tyr3 and Asn5 in the lower half with their side chains pointing downward and approaching the hydrophobic core, Tyr2, Gly4 and His8 and 12 in the upper half, Lys1 near the middle of the interface region, and the side chain of Lys11 pointing out toward the membrane surface.
Keywords: Abbreviations; AMP; antimicrobial peptide; MIC; minimal inhibitory concentration; NMR; nuclear magnetic resonance; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; rac; -glycerol; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholineBacteriocin; Antimicrobial peptide; Peptide–membrane interaction; Sakacin P; Mutagenesis
An antifungal peptide from passion fruit ( Passiflora edulis) seeds with similarities to 2S albumin proteins by P.B. Pelegrini; E.F. Noronha; M.A.R. Muniz; I.M. Vasconcelos; M.D. Chiarello; J.T.A. Oliveira; O.L. Franco (pp. 1141-1146).
An actual worldwide problem consists of an expressive increase of economic losses and health problems caused by fungi. In order to solve this problem, several studies have been concentrating on the screening of novel plant defence peptides with antifungal activities. These peptides are commonly characterized by having low molecular masses and cationic charges. This present work reports on the purification and characterization of a novel plant peptide of 5.0 kDa, Pe-AFP1, purified from the seeds of passion fruit ( Passiflora edulis). Purification was achieved using a Red-Sepharose Cl-6B affinity column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Pe-AFP1 was able of inhibiting the development of the filamentous fungi Trichoderma harzianum, Fusarium oxysporum, and Aspergillus fumigatus with IC50 values of 32, 34, and 40 μg ml−1, respectively, but not of Rhyzoctonia solani, Paracoccidioides brasiliensis and Candida albicans. This protein was also subjected to automated N-terminal amino acid sequence, showing high degree of similarities to storage 2S albumins, adding a new member to this protein-defence family. The discovery of Pe-AFP1 could contribute, in a near future, to the development of biotechnological products as antifungal drugs and transgenic plants with enhanced resistance to pathogenic fungi.
Keywords: Abbreviations; Cp-thionins; thionins from cowpea; CW-1; cheeseweed antifungal protein; HPLC; high-performance liquid chromatography; LTP; lipid-transfer proteins; MYG; malt yeast glucose medium; NaD1; antimicrobial peptide from; Nicotiana alata; Pe; -AFP1; antifungal peptide from; Passiflora edulis; Pp; -AMP1; antimicrobial peptide from; Pyrularia pubera; 1; Pp; -AMP2; antimicrobial peptide from; Pyrularia pubera; 2; PR protein; pathogen-related proteins; Ra-AFP2; Raphanus sativus; antifungal protein isotype 2; RIP; ribosome-inactivating proteins; SDS-PAGE; sodium dodecyl polyacrylamide gel electrophoresis; TFA; trifluoroacetic acid; TL; thaumatin-like; Tu-AMP1; antimicrobial peptide from; Tulipa gesneriana; 1; Tu-AMP2; antimicrobial peptide from; Tulipa gesneriana; 2; VrD1; antimicrobial peptide from; Vigna angularisPlant defence; Antifungal; Passiflora edulis; Pe; -AFP1; 2S albumin; Storage proteins
An antifungal peptide from passion fruit ( Passiflora edulis) seeds with similarities to 2S albumin proteins by P.B. Pelegrini; E.F. Noronha; M.A.R. Muniz; I.M. Vasconcelos; M.D. Chiarello; J.T.A. Oliveira; O.L. Franco (pp. 1141-1146).
An actual worldwide problem consists of an expressive increase of economic losses and health problems caused by fungi. In order to solve this problem, several studies have been concentrating on the screening of novel plant defence peptides with antifungal activities. These peptides are commonly characterized by having low molecular masses and cationic charges. This present work reports on the purification and characterization of a novel plant peptide of 5.0 kDa, Pe-AFP1, purified from the seeds of passion fruit ( Passiflora edulis). Purification was achieved using a Red-Sepharose Cl-6B affinity column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Pe-AFP1 was able of inhibiting the development of the filamentous fungi Trichoderma harzianum, Fusarium oxysporum, and Aspergillus fumigatus with IC50 values of 32, 34, and 40 μg ml−1, respectively, but not of Rhyzoctonia solani, Paracoccidioides brasiliensis and Candida albicans. This protein was also subjected to automated N-terminal amino acid sequence, showing high degree of similarities to storage 2S albumins, adding a new member to this protein-defence family. The discovery of Pe-AFP1 could contribute, in a near future, to the development of biotechnological products as antifungal drugs and transgenic plants with enhanced resistance to pathogenic fungi.
Keywords: Abbreviations; Cp-thionins; thionins from cowpea; CW-1; cheeseweed antifungal protein; HPLC; high-performance liquid chromatography; LTP; lipid-transfer proteins; MYG; malt yeast glucose medium; NaD1; antimicrobial peptide from; Nicotiana alata; Pe; -AFP1; antifungal peptide from; Passiflora edulis; Pp; -AMP1; antimicrobial peptide from; Pyrularia pubera; 1; Pp; -AMP2; antimicrobial peptide from; Pyrularia pubera; 2; PR protein; pathogen-related proteins; Ra-AFP2; Raphanus sativus; antifungal protein isotype 2; RIP; ribosome-inactivating proteins; SDS-PAGE; sodium dodecyl polyacrylamide gel electrophoresis; TFA; trifluoroacetic acid; TL; thaumatin-like; Tu-AMP1; antimicrobial peptide from; Tulipa gesneriana; 1; Tu-AMP2; antimicrobial peptide from; Tulipa gesneriana; 2; VrD1; antimicrobial peptide from; Vigna angularisPlant defence; Antifungal; Passiflora edulis; Pe; -AFP1; 2S albumin; Storage proteins