Carlos Genzor - Academia.edu (original) (raw)
Papers by Carlos Genzor
Biochemistry, 2001
The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site... more The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient intermediate accumulates during the folding reaction. The intermediate is monomeric, and it is not related to proline isomerization. Unlike many cases where the presence of an intermediate has been detected either by a burst phase or by the curvature, at low urea concentration, of the otherwise only observable kinetic phase, two kinetic phases are observed in apoflavodoxin folding whose total amplitude equals the amplitude of unfolding. To determine the role of the intermediate in the folding reaction, the apoflavodoxin kinetic data have been fitted to all conceivable three-species kinetic models (either linear or triangular). Using a stepwise fitting procedure, we find that the off-pathway mechanism explains most of the kinetic data (not a slow unfolding phase), the on-pathway mechanism being rejected. By using global analysis, good overall agreement between data and fit is found when a triangular mechanism is considered. The fitted values of the microscopic constants indicate that most of the unfolded molecules refold from the denatured state. Apoflavodoxin thus folds via a triangular, but essentially off-pathway, mechanism. We calculate that the retardation of the folding caused by the off-pathway intermediate is not large. Some unusual properties of the intermediate are discussed.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1997
Nature Structural Biology, 1996
nature.com homepage Login. Nature Structural & Molecular Biology homepage: My acc... more nature.com homepage Login. Nature Structural & Molecular Biology homepage: My account; E-alert sign up. Register; Subscribe. Publications AZ index; Browse by subject. Search This journal Advanced search. Advertisement. ...
Protein Science, 1996
Flavodoxins are ar/p proteins that mediate electron transfer reactions. The conformational stabil... more Flavodoxins are ar/p proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from A n a b~n a PCC 71 19 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 1 1 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 'H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a prornising system for the analysis of the stability and folding of cr/0 proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.
Clinical Gastroenterology and Hepatology, 2004
We investigated two 1-step immunochromatographic visual assays based on human recombinant tissue-... more We investigated two 1-step immunochromatographic visual assays based on human recombinant tissue-transglutaminase (t-TG) as an alternative to enzyme-linked immunosorbent assays (ELISAs) for celiac disease (CD) screening. We used a tissue-transglutaminase (t-TG) stick, which detected immunoglobulin A/G (IgA/G) antibodies to t-TG, and a t-TG/antigliadin antibodies (AGA) stick, which detected IgA antibodies to both t-TG and AGA, as well as t-TG and AGA ELISAs, to determine t-TG and AGA antibodies in untreated celiac patients with subtotal villous atrophy. A total of 142 children (3 IgA-deficient sera) and 30 adults, and 140 controls (normal mucosa; 121 children and 19 adults), plus 23 sera from pediatric CD patients in remission were assayed. For pediatric patients, with the t-TG stick we obtained a sensitivity of 97.1% and a specificity of 99.0%, and in adults, 83.3% and 100%, respectively. The t-TG/AGA stick displayed a sensitivity of 95.7% and a specificity of 99.0% for t-TG and a sensitivity of 89.2% and a specificity of 95.8% for AGA in children, and a sensitivity of 80% and a specificity of 100% for t-TG and a sensitivity of 83.3% and a specificity of 100% for AGA in adults. Results were comparable with the corresponding ELISAs. The 2 visual assays are efficient for CD screening as an alternative to ELISAs. They are simple to handle and to interpret. By the combined use of the 2 sticks, IgA-deficient patients can be identified as positive in the t-TG (IgG/A) but negative in the t-TG/AGA (IgA) stick.
Biochemistry, 1997
The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ re... more The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd. This is a consequence of oxygen replacing one of the ligating cysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophore. Each mutant is able to rapidly accept an electron from deazariboflavin semiquinone (dRfH.) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantly altered from that of wt Fd (-384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demonstrating that the cluster potential is sensitive to mutations made at the ferric iron in reduced [2Fe-2S] Fds with localized valences. Despite the decrease in thermodynamic driving force for et from C49S to FNR, the et rate constant is similar to that measured for C46S. Thus, the et reactivity of the mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fdred:FNRox) complexes or with the ability of the prosthetic group to be reduced by dRfH.. Furthermore, the X-ray crystal structure of the C49S mutant is virtually identical to that of wt Fd. We conclude from these data that cysteine sulfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutants toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in the electronic structure of the [2Fe-2S] cluster.
Archives of Biochemistry and Biophysics, 1996
A series of charge reversal mutations in a highly conserved acidic patch on the surface of Anabae... more A series of charge reversal mutations in a highly conserved acidic patch on the surface of Anabaena ferredoxin (Fd), comprising residues D67, D68, and D69, have been constructed by site-directed mutagenesis. One such mutant, D68K, has a rate constant for electron transfer (et) to Anabaena ferredoxin:NADP+ reductase (FNR) at low ionic strength (I = 12 mM) which is 2.5 times larger than wild type (9000 vs 3600 s-1). This mutant Fd became indistinguishable from the wild-type protein in its reactivity at I > or = 100 mM. The other mutants showed various degrees of impairment in their et reactions with FNR over the entire range of ionic strengths. The degrees of such impairment for the D67K and D69K mutants were similar to that of the double mutant D67K/D69K. The double mutant D68K/ D69K had et activity intermediate between these mutants and wild type, whereas incorporation of the "super" mutation, D68K, into the double mutant, resulting in the D67K/D68K/D69K triple mutant, did not significantly alter the impairment caused by the D67K/D69K double mutation. Binding constants for complex formation (Kd) between the oxidized mutant proteins and oxidized FNR (except for that of the triple mutant which was not measurable), and the kinetically determined Kd values for the intermediate Fdred:FNRox complex, showed no correlation with et rate constants or with the extent of charge reversal. These results indicate that hydrophobic interactions play a key role in determining complex stability. They also provide strong support for the contention that the specific protein/protein geometry within the Fdred:FNRox intermediate complex is the major determinant of the et rate constants in this series of mutants, and that this is optimized largely by hydrophobic rather than electrostatic interactions. When electrostatic forces are dominant, as they are at low ionic strength, this can lead to nonoptimal et orientations.
Archives of Biochemistry and Biophysics, 1995
Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavo... more Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavodoxin (Fld) modified at surface residues close to the prosthetic groups, with photoexcited P700 in spinach photosystem I (PSI) particles, ferredoxin:NADP+ reductase (FNR), and horse cytochrome c (cytc), have been investigated by laser flash photolysis and stopped-how spectrophotometry. Nonconservative mutations in Fd at F65 and E94, which have been
Biochimie, 1998
Biochimie ~,i998) 80, 813-820 © SociEt6 franq~fise de biochimie el biologic moleculaire / Elsevie... more Biochimie ~,i998) 80, 813-820 © SociEt6 franq~fise de biochimie el biologic moleculaire / Elsevier, Paris Abstract ~ Flavodoxins are one domain a/[~ electron transfer proteins that participate in photosynthetic reactions. All flavodoxins carry a molecule of flavin mononucleotide (FMN), non-covalently bound, that cont~rs redox properties to the protein. There are two structurally distinct flavodoxins, short ones and long flavodoxins; the latter contain an extra loop with unknown function. We have undertaken the study of the stability and folding of the apoflavodoxin from Anabaena (a long flavodoxin) and the analysis of the interaction between the apollavodoxin and FMN. Our studies indicate that apoflavodoxin folds in a few seconds to a form th=~ i.; competent in FMN binding. The stability of this apoflavodoxin is low and its urea denaturation can be described by a :wo-state mechanism. The role of the different parts of the apoflavodoxin in the stability and structure of the whole protein is being investigated using mutagenesis and specific cleavage to generate apoflavodoxin fragments, The X-ray structure of apofiavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex. In apoflavodoxin these groups interact with each other so closing the FMN binding site. Despite this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability. We have found that one role of the aromatic residues that interact with FMN is to help to retain bound the reduced form of the cofactor whose complex with apoflavodoxin is otherwise too weak. © Soci6t6 franqaise de biochimie el biologic molfculaire / Elsevier, Paris fiavodoxin I protein stability I redox potential / protein ligand interaction
Biochemistry, 2001
The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site... more The folding reaction of Anabaena apoflavodoxin has been studied by stopped-flow kinetics and site-directed mutagenesis. Although the urea unfolding equilibrium is two-state, a transient intermediate accumulates during the folding reaction. The intermediate is monomeric, and it is not related to proline isomerization. Unlike many cases where the presence of an intermediate has been detected either by a burst phase or by the curvature, at low urea concentration, of the otherwise only observable kinetic phase, two kinetic phases are observed in apoflavodoxin folding whose total amplitude equals the amplitude of unfolding. To determine the role of the intermediate in the folding reaction, the apoflavodoxin kinetic data have been fitted to all conceivable three-species kinetic models (either linear or triangular). Using a stepwise fitting procedure, we find that the off-pathway mechanism explains most of the kinetic data (not a slow unfolding phase), the on-pathway mechanism being rejected. By using global analysis, good overall agreement between data and fit is found when a triangular mechanism is considered. The fitted values of the microscopic constants indicate that most of the unfolded molecules refold from the denatured state. Apoflavodoxin thus folds via a triangular, but essentially off-pathway, mechanism. We calculate that the retardation of the folding caused by the off-pathway intermediate is not large. Some unusual properties of the intermediate are discussed.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1997
Nature Structural Biology, 1996
nature.com homepage Login. Nature Structural & Molecular Biology homepage: My acc... more nature.com homepage Login. Nature Structural & Molecular Biology homepage: My account; E-alert sign up. Register; Subscribe. Publications AZ index; Browse by subject. Search This journal Advanced search. Advertisement. ...
Protein Science, 1996
Flavodoxins are ar/p proteins that mediate electron transfer reactions. The conformational stabil... more Flavodoxins are ar/p proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from A n a b~n a PCC 71 19 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 1 1 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 'H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a prornising system for the analysis of the stability and folding of cr/0 proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.
Clinical Gastroenterology and Hepatology, 2004
We investigated two 1-step immunochromatographic visual assays based on human recombinant tissue-... more We investigated two 1-step immunochromatographic visual assays based on human recombinant tissue-transglutaminase (t-TG) as an alternative to enzyme-linked immunosorbent assays (ELISAs) for celiac disease (CD) screening. We used a tissue-transglutaminase (t-TG) stick, which detected immunoglobulin A/G (IgA/G) antibodies to t-TG, and a t-TG/antigliadin antibodies (AGA) stick, which detected IgA antibodies to both t-TG and AGA, as well as t-TG and AGA ELISAs, to determine t-TG and AGA antibodies in untreated celiac patients with subtotal villous atrophy. A total of 142 children (3 IgA-deficient sera) and 30 adults, and 140 controls (normal mucosa; 121 children and 19 adults), plus 23 sera from pediatric CD patients in remission were assayed. For pediatric patients, with the t-TG stick we obtained a sensitivity of 97.1% and a specificity of 99.0%, and in adults, 83.3% and 100%, respectively. The t-TG/AGA stick displayed a sensitivity of 95.7% and a specificity of 99.0% for t-TG and a sensitivity of 89.2% and a specificity of 95.8% for AGA in children, and a sensitivity of 80% and a specificity of 100% for t-TG and a sensitivity of 83.3% and a specificity of 100% for AGA in adults. Results were comparable with the corresponding ELISAs. The 2 visual assays are efficient for CD screening as an alternative to ELISAs. They are simple to handle and to interpret. By the combined use of the 2 sticks, IgA-deficient patients can be identified as positive in the t-TG (IgG/A) but negative in the t-TG/AGA (IgA) stick.
Biochemistry, 1997
The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ re... more The reduction potentials and the rate constants for electron transfer (et) to ferredoxin:NADP+ reductase (FNR) are reported for site-directed mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anabaena PCC 7120, each of which has a cluster ligating cysteine residue mutated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (wt) Fd. This is a consequence of oxygen replacing one of the ligating cysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophore. Each mutant is able to rapidly accept an electron from deazariboflavin semiquinone (dRfH.) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantly altered from that of wt Fd (-384 mV), the potential of the C49S mutant (-329 mV) is shifted positively by 55 mV, demonstrating that the cluster potential is sensitive to mutations made at the ferric iron in reduced [2Fe-2S] Fds with localized valences. Despite the decrease in thermodynamic driving force for et from C49S to FNR, the et rate constant is similar to that measured for C46S. Thus, the et reactivity of the mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fdred:FNRox) complexes or with the ability of the prosthetic group to be reduced by dRfH.. Furthermore, the X-ray crystal structure of the C49S mutant is virtually identical to that of wt Fd. We conclude from these data that cysteine sulfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutants toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in the electronic structure of the [2Fe-2S] cluster.
Archives of Biochemistry and Biophysics, 1996
A series of charge reversal mutations in a highly conserved acidic patch on the surface of Anabae... more A series of charge reversal mutations in a highly conserved acidic patch on the surface of Anabaena ferredoxin (Fd), comprising residues D67, D68, and D69, have been constructed by site-directed mutagenesis. One such mutant, D68K, has a rate constant for electron transfer (et) to Anabaena ferredoxin:NADP+ reductase (FNR) at low ionic strength (I = 12 mM) which is 2.5 times larger than wild type (9000 vs 3600 s-1). This mutant Fd became indistinguishable from the wild-type protein in its reactivity at I > or = 100 mM. The other mutants showed various degrees of impairment in their et reactions with FNR over the entire range of ionic strengths. The degrees of such impairment for the D67K and D69K mutants were similar to that of the double mutant D67K/D69K. The double mutant D68K/ D69K had et activity intermediate between these mutants and wild type, whereas incorporation of the "super" mutation, D68K, into the double mutant, resulting in the D67K/D68K/D69K triple mutant, did not significantly alter the impairment caused by the D67K/D69K double mutation. Binding constants for complex formation (Kd) between the oxidized mutant proteins and oxidized FNR (except for that of the triple mutant which was not measurable), and the kinetically determined Kd values for the intermediate Fdred:FNRox complex, showed no correlation with et rate constants or with the extent of charge reversal. These results indicate that hydrophobic interactions play a key role in determining complex stability. They also provide strong support for the contention that the specific protein/protein geometry within the Fdred:FNRox intermediate complex is the major determinant of the et rate constants in this series of mutants, and that this is optimized largely by hydrophobic rather than electrostatic interactions. When electrostatic forces are dominant, as they are at low ionic strength, this can lead to nonoptimal et orientations.
Archives of Biochemistry and Biophysics, 1995
Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavo... more Electron transfer reactions involving site-specific mutants of Anabaena ferredoxin (Fd) and flavodoxin (Fld) modified at surface residues close to the prosthetic groups, with photoexcited P700 in spinach photosystem I (PSI) particles, ferredoxin:NADP+ reductase (FNR), and horse cytochrome c (cytc), have been investigated by laser flash photolysis and stopped-how spectrophotometry. Nonconservative mutations in Fd at F65 and E94, which have been
Biochimie, 1998
Biochimie ~,i998) 80, 813-820 © SociEt6 franq~fise de biochimie el biologic moleculaire / Elsevie... more Biochimie ~,i998) 80, 813-820 © SociEt6 franq~fise de biochimie el biologic moleculaire / Elsevier, Paris Abstract ~ Flavodoxins are one domain a/[~ electron transfer proteins that participate in photosynthetic reactions. All flavodoxins carry a molecule of flavin mononucleotide (FMN), non-covalently bound, that cont~rs redox properties to the protein. There are two structurally distinct flavodoxins, short ones and long flavodoxins; the latter contain an extra loop with unknown function. We have undertaken the study of the stability and folding of the apoflavodoxin from Anabaena (a long flavodoxin) and the analysis of the interaction between the apollavodoxin and FMN. Our studies indicate that apoflavodoxin folds in a few seconds to a form th=~ i.; competent in FMN binding. The stability of this apoflavodoxin is low and its urea denaturation can be described by a :wo-state mechanism. The role of the different parts of the apoflavodoxin in the stability and structure of the whole protein is being investigated using mutagenesis and specific cleavage to generate apoflavodoxin fragments, The X-ray structure of apofiavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex. In apoflavodoxin these groups interact with each other so closing the FMN binding site. Despite this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability. We have found that one role of the aromatic residues that interact with FMN is to help to retain bound the reduced form of the cofactor whose complex with apoflavodoxin is otherwise too weak. © Soci6t6 franqaise de biochimie el biologic molfculaire / Elsevier, Paris fiavodoxin I protein stability I redox potential / protein ligand interaction