The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Ewan J Mcadam - One of the best experts on this subject based on the ideXlab platform.
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controlling shell Side crystal nucleation in a gas liquid membrane contactor for simultaneous ammonium bicarbonate recovery and biogas upgrading
Journal of Membrane Science, 2015Co-Authors: Andrew J Mcleod, P Buzatu, Olivier Autin, Bruce Jefferson, Ewan J McadamAbstract:Abstract A gas–liquid hollow fibre membrane contactor (HFMC) process has been introduced for carbon dioxide (CO2) separation from biogas where aqueous ammonia (NH3) is used to chemically enhance CO2 absorption and initiate heterogeneous nucleation of the reaction product ammonium bicarbonate at the membrane–solvent interface. Aqueous ammonia absorbents (2–7 M) were initially used in single pass for CO2 separation from a synthetic biogas where nucleation of ammonium bicarbonate crystals was observed at the perimeter of the micropores. Recirculation of the aqueous ammonia absorbent encouraged the growth of ammonium bicarbonate crystals on the shell-Side of the membrane that measured several microns in diameter. However, at high aqueous NH3 concentrations (3–7 M), Lumen Side crystallisation occurred and obstructed gas flow through the Lumen of the HFMC. The suggested mechanism for Lumen-Side crystallisation was absorbent breakthrough into the Lumen due to pore wetting which was promoted by low absorbent surface tension at high NH3 concentration. Preferential shell-Side nucleation can therefore be promoted by (1) raising surface tension of the absorbent and (2) selection of a membrane with a more regulated pore shape than the PTFE membrane used (d/L 0.065) as both actions can diminish solvent ingress into the pore. This was evidenced using 2 M NH3 absorbent where shell-Side crystallisation was evidenced without the onset of Lumen Side crystallisation. Raising surface tension through the inclusion of salt into the chemical absorbent also promoted greater CO2 flux stability. Importantly, this study demonstrates that chemically enhanced HFMC are an attractive prospect for gas–liquid separation applications where reaction product recovery offers further economic value.
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Controlling shell-Side crystal nucleation in a gas–liquid membrane contactor for simultaneous ammonium bicarbonate recovery and biogas upgrading
Journal of Membrane Science, 2015Co-Authors: Andrew J Mcleod, P Buzatu, Olivier Autin, Bruce Jefferson, Ewan J McadamAbstract:Abstract A gas–liquid hollow fibre membrane contactor (HFMC) process has been introduced for carbon dioxide (CO2) separation from biogas where aqueous ammonia (NH3) is used to chemically enhance CO2 absorption and initiate heterogeneous nucleation of the reaction product ammonium bicarbonate at the membrane–solvent interface. Aqueous ammonia absorbents (2–7 M) were initially used in single pass for CO2 separation from a synthetic biogas where nucleation of ammonium bicarbonate crystals was observed at the perimeter of the micropores. Recirculation of the aqueous ammonia absorbent encouraged the growth of ammonium bicarbonate crystals on the shell-Side of the membrane that measured several microns in diameter. However, at high aqueous NH3 concentrations (3–7 M), Lumen Side crystallisation occurred and obstructed gas flow through the Lumen of the HFMC. The suggested mechanism for Lumen-Side crystallisation was absorbent breakthrough into the Lumen due to pore wetting which was promoted by low absorbent surface tension at high NH3 concentration. Preferential shell-Side nucleation can therefore be promoted by (1) raising surface tension of the absorbent and (2) selection of a membrane with a more regulated pore shape than the PTFE membrane used (d/L 0.065) as both actions can diminish solvent ingress into the pore. This was evidenced using 2 M NH3 absorbent where shell-Side crystallisation was evidenced without the onset of Lumen Side crystallisation. Raising surface tension through the inclusion of salt into the chemical absorbent also promoted greater CO2 flux stability. Importantly, this study demonstrates that chemically enhanced HFMC are an attractive prospect for gas–liquid separation applications where reaction product recovery offers further economic value.
Janet L. Smith - One of the best experts on this subject based on the ideXlab platform.
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biological identity and diversity in photosynthesis and respiration structure of the Lumen Side domain of the chloroplast rieske protein
Structure, 1997Co-Authors: Christopher J. Carrell, William A. Cramer, Huamin Zhang, Janet L. SmithAbstract:Abstract Background: The cytochrome b 6 f complex functions in oxygenic photosynthesis as an integral membrane protein complex that mediates coupled electron transfer and proton translocation. The Rieske [2Fe–2S] protein subunit of the complex functions at the electropositive ( p ) membrane interface as the electron acceptor for plastoquinol and donor for the cytochrome f subunit, and may have a dynamic role in catalyzing electron and proton transfer at the membrane interface. There are significant structure/function similarities to the cytochrome bc 1 complex of the respiratory chain. Results: The 1.83 A crystal structure of a 139-residue C-terminal fragment of the Rieske [2Fe–2S] protein, derived from the cytochrome b 6 f complex of spinach chloroplasts, has been solved by multiwavelength anomalous diffraction. The structure of the fragment comprises two domains: a small ‘cluster-binding' subdomain and a large subdomain. The [2Fe–2S] cluster-binding subdomains of the chloroplast and mitochondrial Rieske proteins are virtually identical, whereas the large subdomains are strikingly different despite a common folding topology. A structure-based sequence alignment of the b 6 f and bc 1 groups of Rieske soluble domains is presented. Conclusions: The segregation of structural conservation and divergence in the cluster-binding and large subdomains of the Rieske protein correlates with the overall relatedness of the cytochrome b 6 f and bc 1 complexes, in which redox domains in the aqueous p phase are dissimilar and those within the membrane are similar. Distinct sequences and surface charge distributions among Rieske large subdomains may provide a signature for interaction with the p -Side oxidant protein and for the pH of the intraorganelle compartment.
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Characterization and crystallization of the Lumen Side domain of the chloroplast Rieske iron-sulfur protein.
The Journal of biological chemistry, 1996Co-Authors: Huamin Zhang, Janet L. Smith, D Huang, Christopher J. Carrell, Vladimir Sled, Tomoko Ohnishi, William A. CramerAbstract:A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b6f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a two-step chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 ± 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by ΔϵmM = 3.8 mM−1 cm−1 for ΔA at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is +359 ± 7 mV at 25°C from pH 5.5-6.5, and +319 ± 2 mV at pH 7, with an apparent pKox = 6.5 ± 0.2 for the oxidized protein. The EPR spectrum measured at 17 K was characterized by the g values, gz = 2.03 and gy = 1.90, and a broad band centered at gx≈ 1.74, very similar or identical to those of the Rieske cluster in the b6f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex. Midpoint potential determination by low temperature EPR yielded a redox midpoint potential (Em) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an Em of +295-300 mV of the Rieske cluster in the cytochrome b6f complex at pH 6 and 7. The Em difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex. Single crystals of the Rieske polypeptide were obtained that are capable of x-ray diffraction to atomic resolution (
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the heme redox center of chloroplast cytochrome f is linked to a buried five water chain
Protein Science, 1996Co-Authors: Sergio E Martinez, William A. Cramer, D Huang, M Ponomarev, Janet L. SmithAbstract:The crystal structure of the 252-residue Lumen-Side domain of reduced cytochrome f, a subunit of the proton-pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35 degrees. The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20 degrees, the structure at -35 degrees has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L-shaped array of five buried water molecules that extend in two directions from the N delta 1 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the Lumen-Side exit port for proton translocation by the cytochrome b6f complex.
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The heme redox center of chloroplast cytochrome f is linked to a buried five‐water chain
Protein science : a publication of the Protein Society, 1996Co-Authors: Sergio E Martinez, William A. Cramer, D Huang, M Ponomarev, Janet L. SmithAbstract:The crystal structure of the 252-residue Lumen-Side domain of reduced cytochrome f, a subunit of the proton-pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35 degrees. The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20 degrees, the structure at -35 degrees has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L-shaped array of five buried water molecules that extend in two directions from the N delta 1 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the Lumen-Side exit port for proton translocation by the cytochrome b6f complex.
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Structural aspects of the cytochrome b6f complex; structure of the Lumen-Side domain of cytochrome f
Journal of bioenergetics and biomembranes, 1994Co-Authors: William A. Cramer, Sergio E Martinez, D Huang, G. S. Tae, R. M. Everly, J. B. Heymann, R. H. Cheng, Timothy S. Baker, Janet L. SmithAbstract:The following findings concerning the structure of the cytochromeb6f complex and its component polypeptides, cytb6, subunit IV and cytochromef subunit are discussed: (1) Comparison of the amino acid sequences of 13 and 16 cytochromeb6 and subunit IV polypeptides, respectively, led to (a) reconSideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cytb6 and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudoconserved residues) in the segments of cytb6 predicted to be extrinsic on then-Side of the membrane. (2) The intramembrane attractive forces betweentrans-membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. (3) The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. (4) The isolated cytb6f complex contains one molecule of chlorophylla. (5) The structure of the 252 residue Lumen-Side domain of cytochromef isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 A.
William A. Cramer - One of the best experts on this subject based on the ideXlab platform.
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biological identity and diversity in photosynthesis and respiration structure of the Lumen Side domain of the chloroplast rieske protein
Structure, 1997Co-Authors: Christopher J. Carrell, William A. Cramer, Huamin Zhang, Janet L. SmithAbstract:Abstract Background: The cytochrome b 6 f complex functions in oxygenic photosynthesis as an integral membrane protein complex that mediates coupled electron transfer and proton translocation. The Rieske [2Fe–2S] protein subunit of the complex functions at the electropositive ( p ) membrane interface as the electron acceptor for plastoquinol and donor for the cytochrome f subunit, and may have a dynamic role in catalyzing electron and proton transfer at the membrane interface. There are significant structure/function similarities to the cytochrome bc 1 complex of the respiratory chain. Results: The 1.83 A crystal structure of a 139-residue C-terminal fragment of the Rieske [2Fe–2S] protein, derived from the cytochrome b 6 f complex of spinach chloroplasts, has been solved by multiwavelength anomalous diffraction. The structure of the fragment comprises two domains: a small ‘cluster-binding' subdomain and a large subdomain. The [2Fe–2S] cluster-binding subdomains of the chloroplast and mitochondrial Rieske proteins are virtually identical, whereas the large subdomains are strikingly different despite a common folding topology. A structure-based sequence alignment of the b 6 f and bc 1 groups of Rieske soluble domains is presented. Conclusions: The segregation of structural conservation and divergence in the cluster-binding and large subdomains of the Rieske protein correlates with the overall relatedness of the cytochrome b 6 f and bc 1 complexes, in which redox domains in the aqueous p phase are dissimilar and those within the membrane are similar. Distinct sequences and surface charge distributions among Rieske large subdomains may provide a signature for interaction with the p -Side oxidant protein and for the pH of the intraorganelle compartment.
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Characterization and crystallization of the Lumen Side domain of the chloroplast Rieske iron-sulfur protein.
The Journal of biological chemistry, 1996Co-Authors: Huamin Zhang, Janet L. Smith, D Huang, Christopher J. Carrell, Vladimir Sled, Tomoko Ohnishi, William A. CramerAbstract:A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b6f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a two-step chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 ± 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by ΔϵmM = 3.8 mM−1 cm−1 for ΔA at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is +359 ± 7 mV at 25°C from pH 5.5-6.5, and +319 ± 2 mV at pH 7, with an apparent pKox = 6.5 ± 0.2 for the oxidized protein. The EPR spectrum measured at 17 K was characterized by the g values, gz = 2.03 and gy = 1.90, and a broad band centered at gx≈ 1.74, very similar or identical to those of the Rieske cluster in the b6f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex. Midpoint potential determination by low temperature EPR yielded a redox midpoint potential (Em) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an Em of +295-300 mV of the Rieske cluster in the cytochrome b6f complex at pH 6 and 7. The Em difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex. Single crystals of the Rieske polypeptide were obtained that are capable of x-ray diffraction to atomic resolution (
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the heme redox center of chloroplast cytochrome f is linked to a buried five water chain
Protein Science, 1996Co-Authors: Sergio E Martinez, William A. Cramer, D Huang, M Ponomarev, Janet L. SmithAbstract:The crystal structure of the 252-residue Lumen-Side domain of reduced cytochrome f, a subunit of the proton-pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35 degrees. The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20 degrees, the structure at -35 degrees has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L-shaped array of five buried water molecules that extend in two directions from the N delta 1 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the Lumen-Side exit port for proton translocation by the cytochrome b6f complex.
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The heme redox center of chloroplast cytochrome f is linked to a buried five‐water chain
Protein science : a publication of the Protein Society, 1996Co-Authors: Sergio E Martinez, William A. Cramer, D Huang, M Ponomarev, Janet L. SmithAbstract:The crystal structure of the 252-residue Lumen-Side domain of reduced cytochrome f, a subunit of the proton-pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35 degrees. The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20 degrees, the structure at -35 degrees has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L-shaped array of five buried water molecules that extend in two directions from the N delta 1 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the Lumen-Side exit port for proton translocation by the cytochrome b6f complex.
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Structural aspects of the cytochrome b6f complex; structure of the Lumen-Side domain of cytochrome f
Journal of bioenergetics and biomembranes, 1994Co-Authors: William A. Cramer, Sergio E Martinez, D Huang, G. S. Tae, R. M. Everly, J. B. Heymann, R. H. Cheng, Timothy S. Baker, Janet L. SmithAbstract:The following findings concerning the structure of the cytochromeb6f complex and its component polypeptides, cytb6, subunit IV and cytochromef subunit are discussed: (1) Comparison of the amino acid sequences of 13 and 16 cytochromeb6 and subunit IV polypeptides, respectively, led to (a) reconSideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cytb6 and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudoconserved residues) in the segments of cytb6 predicted to be extrinsic on then-Side of the membrane. (2) The intramembrane attractive forces betweentrans-membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. (3) The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. (4) The isolated cytb6f complex contains one molecule of chlorophylla. (5) The structure of the 252 residue Lumen-Side domain of cytochromef isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 A.
Andrew J Mcleod - One of the best experts on this subject based on the ideXlab platform.
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controlling shell Side crystal nucleation in a gas liquid membrane contactor for simultaneous ammonium bicarbonate recovery and biogas upgrading
Journal of Membrane Science, 2015Co-Authors: Andrew J Mcleod, P Buzatu, Olivier Autin, Bruce Jefferson, Ewan J McadamAbstract:Abstract A gas–liquid hollow fibre membrane contactor (HFMC) process has been introduced for carbon dioxide (CO2) separation from biogas where aqueous ammonia (NH3) is used to chemically enhance CO2 absorption and initiate heterogeneous nucleation of the reaction product ammonium bicarbonate at the membrane–solvent interface. Aqueous ammonia absorbents (2–7 M) were initially used in single pass for CO2 separation from a synthetic biogas where nucleation of ammonium bicarbonate crystals was observed at the perimeter of the micropores. Recirculation of the aqueous ammonia absorbent encouraged the growth of ammonium bicarbonate crystals on the shell-Side of the membrane that measured several microns in diameter. However, at high aqueous NH3 concentrations (3–7 M), Lumen Side crystallisation occurred and obstructed gas flow through the Lumen of the HFMC. The suggested mechanism for Lumen-Side crystallisation was absorbent breakthrough into the Lumen due to pore wetting which was promoted by low absorbent surface tension at high NH3 concentration. Preferential shell-Side nucleation can therefore be promoted by (1) raising surface tension of the absorbent and (2) selection of a membrane with a more regulated pore shape than the PTFE membrane used (d/L 0.065) as both actions can diminish solvent ingress into the pore. This was evidenced using 2 M NH3 absorbent where shell-Side crystallisation was evidenced without the onset of Lumen Side crystallisation. Raising surface tension through the inclusion of salt into the chemical absorbent also promoted greater CO2 flux stability. Importantly, this study demonstrates that chemically enhanced HFMC are an attractive prospect for gas–liquid separation applications where reaction product recovery offers further economic value.
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Controlling shell-Side crystal nucleation in a gas–liquid membrane contactor for simultaneous ammonium bicarbonate recovery and biogas upgrading
Journal of Membrane Science, 2015Co-Authors: Andrew J Mcleod, P Buzatu, Olivier Autin, Bruce Jefferson, Ewan J McadamAbstract:Abstract A gas–liquid hollow fibre membrane contactor (HFMC) process has been introduced for carbon dioxide (CO2) separation from biogas where aqueous ammonia (NH3) is used to chemically enhance CO2 absorption and initiate heterogeneous nucleation of the reaction product ammonium bicarbonate at the membrane–solvent interface. Aqueous ammonia absorbents (2–7 M) were initially used in single pass for CO2 separation from a synthetic biogas where nucleation of ammonium bicarbonate crystals was observed at the perimeter of the micropores. Recirculation of the aqueous ammonia absorbent encouraged the growth of ammonium bicarbonate crystals on the shell-Side of the membrane that measured several microns in diameter. However, at high aqueous NH3 concentrations (3–7 M), Lumen Side crystallisation occurred and obstructed gas flow through the Lumen of the HFMC. The suggested mechanism for Lumen-Side crystallisation was absorbent breakthrough into the Lumen due to pore wetting which was promoted by low absorbent surface tension at high NH3 concentration. Preferential shell-Side nucleation can therefore be promoted by (1) raising surface tension of the absorbent and (2) selection of a membrane with a more regulated pore shape than the PTFE membrane used (d/L 0.065) as both actions can diminish solvent ingress into the pore. This was evidenced using 2 M NH3 absorbent where shell-Side crystallisation was evidenced without the onset of Lumen Side crystallisation. Raising surface tension through the inclusion of salt into the chemical absorbent also promoted greater CO2 flux stability. Importantly, this study demonstrates that chemically enhanced HFMC are an attractive prospect for gas–liquid separation applications where reaction product recovery offers further economic value.
D Huang - One of the best experts on this subject based on the ideXlab platform.
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Characterization and crystallization of the Lumen Side domain of the chloroplast Rieske iron-sulfur protein.
The Journal of biological chemistry, 1996Co-Authors: Huamin Zhang, Janet L. Smith, D Huang, Christopher J. Carrell, Vladimir Sled, Tomoko Ohnishi, William A. CramerAbstract:A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b6f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a two-step chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 ± 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by ΔϵmM = 3.8 mM−1 cm−1 for ΔA at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is +359 ± 7 mV at 25°C from pH 5.5-6.5, and +319 ± 2 mV at pH 7, with an apparent pKox = 6.5 ± 0.2 for the oxidized protein. The EPR spectrum measured at 17 K was characterized by the g values, gz = 2.03 and gy = 1.90, and a broad band centered at gx≈ 1.74, very similar or identical to those of the Rieske cluster in the b6f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex. Midpoint potential determination by low temperature EPR yielded a redox midpoint potential (Em) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an Em of +295-300 mV of the Rieske cluster in the cytochrome b6f complex at pH 6 and 7. The Em difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex. Single crystals of the Rieske polypeptide were obtained that are capable of x-ray diffraction to atomic resolution (
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the heme redox center of chloroplast cytochrome f is linked to a buried five water chain
Protein Science, 1996Co-Authors: Sergio E Martinez, William A. Cramer, D Huang, M Ponomarev, Janet L. SmithAbstract:The crystal structure of the 252-residue Lumen-Side domain of reduced cytochrome f, a subunit of the proton-pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35 degrees. The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20 degrees, the structure at -35 degrees has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L-shaped array of five buried water molecules that extend in two directions from the N delta 1 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the Lumen-Side exit port for proton translocation by the cytochrome b6f complex.
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The heme redox center of chloroplast cytochrome f is linked to a buried five‐water chain
Protein science : a publication of the Protein Society, 1996Co-Authors: Sergio E Martinez, William A. Cramer, D Huang, M Ponomarev, Janet L. SmithAbstract:The crystal structure of the 252-residue Lumen-Side domain of reduced cytochrome f, a subunit of the proton-pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 A from crystals cooled to -35 degrees. The model was refined to an R-factor of 15.8% with a 0.013-A RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20 degrees, the structure at -35 degrees has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L-shaped array of five buried water molecules that extend in two directions from the N delta 1 of the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the Lumen-Side exit port for proton translocation by the cytochrome b6f complex.
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Structural aspects of the cytochromeb _6 f complex; structure of the Lumen-Side domain of cytochromef
Journal of Bioenergetics and Biomembranes, 1994Co-Authors: W. A. Cramer, D Huang, G. S. Tae, R. M. Everly, J. B. Heymann, R. H. Cheng, Timothy S. Baker, S. E. Martinez, J. L. SmithAbstract:The following findings concerning the structure of the cytochrome b _6 f complex and its component polypeptides, cyt b _6, subunit IV and cytochrome f subunit are discussed: (1) Comparison of the amino acid sequences of 13 and 16 cytochrome b _6 and subunit IV polypeptides, respectively, led to (a) reconSideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cyt b _6 and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudoconserved residues) in the segments of cyt b _6 predicted to be extrinsic on the n -Side of the membrane. (2) The intramembrane attractive forces between trans -membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. (3) The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. (4) The isolated cyt b _6 f complex contains one molecule of chlorophyll a . (5) The structure of the 252 residue Lumen-Side domain of cytochrome f isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 Å.
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Structural aspects of the cytochrome b6f complex; structure of the Lumen-Side domain of cytochrome f
Journal of bioenergetics and biomembranes, 1994Co-Authors: William A. Cramer, Sergio E Martinez, D Huang, G. S. Tae, R. M. Everly, J. B. Heymann, R. H. Cheng, Timothy S. Baker, Janet L. SmithAbstract:The following findings concerning the structure of the cytochromeb6f complex and its component polypeptides, cytb6, subunit IV and cytochromef subunit are discussed: (1) Comparison of the amino acid sequences of 13 and 16 cytochromeb6 and subunit IV polypeptides, respectively, led to (a) reconSideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cytb6 and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudoconserved residues) in the segments of cytb6 predicted to be extrinsic on then-Side of the membrane. (2) The intramembrane attractive forces betweentrans-membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. (3) The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. (4) The isolated cytb6f complex contains one molecule of chlorophylla. (5) The structure of the 252 residue Lumen-Side domain of cytochromef isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 A.
