Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Ultrasound-assisted MnO2 catalyzed homolysis of peracetic acid for phenol degradation: The assessment of process chemistry and kinetics
Rokhina, E.V. ; Makarova, K. ; Lathinen, M. ; Golovina, E.A. ; As, H. van; Virkutyte, J. - \ 2013
Chemical Engineering Journal 221 (2013). - ISSN 1385-8947 - p. 476 - 486.
wet peroxide oxidation - aqueous-solutions - free-radicals - mechanism - systems - water - decomposition - sonochemistry - intermediate - destruction
The combination of peracetic acid (PAA) and heterogeneous catalyst (MnO2) was used for the degradation of phenol in an aqueous solution in the presence of ultrasound irradiation (US). As a relevant source of free radicals (e.g. OH), peracetic acid was comprehensively studied by means of electron spin resonance (ESR) spin trapping (ST) techniques with the subsequent identification of free radicals by simulation based fitting (SBF) technique. The radical reaction mechanism, where hydroxyl radical was a primary product of OO bond rupture of PAA, was established taking into account radical reactions, occurring during sonolysis. The potential barriers and the reaction heat were determined by basic density function theory (DFT) calculations to estimate whether the proposed radical pathway is possible. The assessment and optimization of the process parameters for MnO2/PAA/US system to eliminate phenol was accomplished with experimental design. Fractional factorial design (FFD) was executed to relate the removal efficiency of phenol with process parameters such as catalyst and PAA concentrations, the presence of ultrasound and the reaction time. The comparative kinetic study of silent and ultrasound-assisted processes revealed the significant difference between these two processes that was mainly attributed to the complex radical system formed during PAA homolysis
Fluorescence of Alexa Fluor dye tracks protein folding
Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Visser, A.J.W.G. ; Mierlo, C.P.M. van - \ 2012
PLoS ONE 7 (2012)10. - ISSN 1932-6203 - 8 p.
azotobacter-vinelandii apoflavodoxin - resonance energy-transfer - beta parallel protein - molten-globule state - flavodoxin-ii - molecules - pathway - chains - intermediate - spectroscopy
Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.
"Noncooperative formation of the off-pathway molten globule during folding of the a-ß parallel protein apoflavodoxin"
Nabuurs, S.M. ; Westphal, A.H. ; Mierlo, C.P.M. van - \ 2009
Journal of the American Chemical Society 131 (2009)7. - ISSN 0002-7863 - p. 2739 - 2746.
azotobacter-vinelandii apoflavodoxin - nmr-spectroscopy - structural-characterization - different denaturants - unfolded molecules - hydrogen-exchange - energy landscape - transition-state - on-pathway - intermediate
During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner
Extensive formation of off-pathway species during folding of an alpha-beta parallel protein is due to docking of (non)native structure elements in unfolded molecules
Nabuurs, S.M. ; Westphal, A.H. ; Mierlo, C.P.M. van - \ 2008
Journal of the American Chemical Society 130 (2008)50. - ISSN 0002-7863 - p. 16914 - 16920.
azotobacter-vinelandii apoflavodoxin - nmr chemical-shifts - 8 m urea - secondary structure - denatured state - hydrogen-exchange - energy landscape - intermediate - topology - conformations
Detailed information about unfolded states is required to understand how proteins fold. Knowledge about folding intermediates formed subsequently is essential to get a grip on pathological aggregation phenomena. During folding of apoflavodoxin, which adopts the widely prevalent ¿¿ß parallel topology, most molecules fold via an off-pathway folding intermediate with helical properties. To better understand why this species is formed, guanidine hydrochloride-unfolded apoflavodoxin is characterized at the residue level using heteronuclear NMR spectroscopy. In 6.0 M denaturant, the protein behaves as a random coil. In contrast, at 3.4 M denaturant, secondary shifts and 1H¿15N relaxation rates report four transiently ordered regions in unfolded apoflavodoxin. These regions have restricted flexibility on the (sub)nanosecond time scale. Secondary shifts show that three of these regions form ¿-helices, which are populated about 10% of the time, as confirmed by far-UV CD data. One region of unfolded apoflavodoxin adopts non-native structure. Of the ¿-helices observed, two are present in native apoflavodoxin as well. A substantial part of the third helix becomes ß-strand while forming native protein. Chemical shift changes due to amino acid residue replacement show that the latter ¿-helix has hydrophobic interactions with all other ordered regions in unfolded apoflavodoxin. Remarkably, these ordered segments dock non-natively, which causes strong competition with on-pathway folding. Thus, rather than directing productive folding, conformational preorganization in the unfolded state of an ¿¿ß parallel-type protein promotes off-pathway species formation.
Tertiary structure in 7.9 M guanidinium chloride: the role of Glu-53 and Asp-287 in Pyrococcus furiosus endo-beta-1,3-glucanase
Chiaraluce, R. ; Florio, R. ; Angelaccio, S. ; Gianese, G. ; Lieshout, J.F.T. van; Oost, J. van der; Consalvi, V. - \ 2007
FEBS Journal 274 (2007). - ISSN 1742-464X - p. 6167 - 6179.
denatured state - staphylococcal nuclease - protein stability - unfolded state - thermophilic proteins - residual structure - m-values - intermediate - thermodynamics - salt
The thermodynamic stability of family 16 endo-ß-1,3-glucanase (EC from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction, both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. The present study shows that, in P. furiosus, endo-ß-1,3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 m guanidinium chloride. The persistence of tertiary structure in 7.9 m guanidinium chloride, a feature of the wild-type enzyme, is observed also for the three variant proteins. The ¿GH2O values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximatelty 50% lower than that of the wild-type. The destabilizing effect of the combined mutations of the double mutant is non-additive, with an energy of interaction of 24.2 kJ·mol¿1, suggesting a communication between the two mutated residues. The decrease in the thermodynamic stability of D287A, E53A and E53A/D287A is contained almost exclusively in the m-values, a parameter which reflects the solvent-exposed surface area upon unfolding. The decrease in m-value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non-native state in 7.9 m guanidinium chloride comparable to that induced by the presence of calcium on the wild-type. These results suggest that the stabilization of a compact non-native state may be a strategy for P. furiosus endo-ß-1,3-glucanase to thrive under adverse environmental conditions.
Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone
Posthuma-Trumpie, G.A. ; Berg, W.A.M. van den; Wiel, D.F.M. van de; Schaaper, W.M.M. ; Korf, J. ; Berkel, W.J.H. van - \ 2007
Biochimica et Biophysica Acta. Proteins & Proteomics 1774 (2007)7. - ISSN 1570-9639 - p. 803 - 812.
flavin-adenine-dinucleotide - apoenzyme reactivation immunoassay - glucose-oxidase - aspergillus-niger - colorimetric immunoassays - enzyme-electrode - binding - label - milk - intermediate
The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50 ± 10%), reconstitutable dimers (20 ± 10%) and irreversibly aggregated oligomers (30 ± 20%). Incubation of monomeric apoGOx with FAD or N6-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N6-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25¿50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.
Calcium-induced tertiary structure modifications of endo-B-1,3-glucanase form Pyrococcus furiosus in 7.9 M guanidinium chloride
Chiaraluce, R. ; Gianese, G. ; Angelaccio, S. ; Florio, R. ; Lieshout, J.F.T. van; Oost, J. van der; Consalvi, V. - \ 2005
Biochemical Journal 386 (2005)3. - ISSN 0264-6021 - p. 515 - 524.
transform infrared-spectroscopy - protein secondary structure - 3-dimensional structures - ftir spectroscopy - free-energy - recognition - database - endo-beta-1,3-glucanase - intermediate - alignments
The family 16 endo-b-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-b-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases
Last in, first out: the role of cofactor binding in flavodoxin folding
Bollen, Y.J.M. ; Nabuurs, S.M. ; Berkel, W.J.H. van; Mierlo, C.P.M. van - \ 2005
Journal of Biological Chemistry 280 (2005)9. - ISSN 0021-9258 - p. 7836 - 7844.
desulfovibrio-desulfuricans flavodoxin - azotobacter-vinelandii apoflavodoxin - mononucleotide cofactor binding - vulgaris flavodoxin - analog binding - kinetics - protein - thermodynamics - reconstitution - intermediate
Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I1 ¿ unfolded apoflavodoxin ¿ I2 ¿ native apoflavodoxin. I1 is an off-pathway molten globule-like intermediate that populates during denaturant-induced equilibrium unfolding; I2 is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I1 + FMN ¿ unfolded apoflavodoxin + FMN ¿ I2 + FMN ¿ native apoflavodoxin + FMN ¿ holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I1, 12, and unfolded apoflavodoxin do not interact to a significant extent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur
Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy
Olofsson, A. ; Ippel, J.H. ; Wijmenga, S.S. ; Lundgren, E. ; Ohman, A. - \ 2004
Journal of Biological Chemistry 279 (2004)7. - ISSN 0021-9258 - p. 5699 - 5707.
hydrogen-exchange - tetramer dissociation - subunit interface - x-ray - fibrils - core - intermediate - proteins - variants - mutants
The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six beta-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F' and H-H' with a prolongation of these beta-strands and, interestingly, with a possible shift in beta-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.
Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions
Dijk, J.A. ; Gerritse, J. ; Schraa, G. ; Stams, A.J.M. - \ 2004
Archives of Microbiology 182 (2004)6. - ISSN 0302-8933 - p. 514 - 519.
vinyl-chloride - pyrroloquinoline quinone - dehydrogenase - metabolism - pqq - mineralization - chloroethanol - intermediate - oxidation - aquifer
The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected
The activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH
Perugino, G. ; Trincone, A. ; Giordano, A. ; Oost, J. van der; Kaper, T. ; Rossi, M. ; Moracci, M. - \ 2003
Biochemistry 42 (2003). - ISSN 0006-2960 - p. 8484 - 8493.
agrobacterium beta-glucosidase - oligosaccharide synthesis - sulfolobus-solfataricus - glycosyl transfer - glycosidase - intermediate - pyrococcus - mechanism - evolution - protein
We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus so fataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the P-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.
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