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Jean-louis Borgna - One of the best experts on this subject based on the ideXlab platform.
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Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor α
The Journal of Steroid Biochemistry and Molecular Biology, 2006Co-Authors: Hélène Mattras, Sigrid Aliau, Emmanuelle Demey, Joël Poncet, Jean-louis BorgnaAbstract:Abstract A purified preparation of human estrogen receptor α (hERα) ligand-binding domain (LBD) involving mainly the Ser309Ala569 (∼30%) and Ser309Ala571 (∼63%) ER portions was used to identify the Covalent Attachment sites of two closely related estrogenic ER affinity labels 17α-bromoacetamidopropylestradiol (17BAPE2) and 17α-bromoacetamidomethylestradiol (17BAME2). To identify and quantify the electrophile Covalent Attachment sites, [14C]17BAPE2- and [14C]17BAME2-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys530Lys531 and Cys417Arg434 hLBD tryptic peptides. The Covalent Attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys530 and Cys417. However, the balance between Cys530 and Cys417 labeling markedly differed according to the affinity label used, with the Cys530/Cys417 ratio being 2.1 for 17BAPE2, and 20 for 17BAME2. We attempted to interpret the Covalent Attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E2. In agreement with the different levels of Cys417 alkylation, the LBD model with unchanged helices could not easily account for Cys417 labeling by 17BAME2, whereas favorable results were obtained through 17BAPE2 docking. Moreover, labeling at Cys530 by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E2 17α-derivatives differ from the structure of crystallized LBD bound to E2.
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Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor alpha.
The Journal of steroid biochemistry and molecular biology, 2006Co-Authors: Hélène Mattras, Sigrid Aliau, Emmanuelle Demey, Joël Poncet, Jean-louis BorgnaAbstract:A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the Covalent Attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile Covalent Attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The Covalent Attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the Covalent Attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).
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Identification of Covalent Attachment site of antiestrogenic estradiol 11β-derivatives on human estrogen receptor α ligand-binding domain
The Journal of Steroid Biochemistry and Molecular Biology, 2006Co-Authors: Sigrid Aliau, Hélène Mattras, Jean-louis BorgnaAbstract:Abstract Affinity labeling of human estrogen receptor α (ERα) by high affinity and antiestrogenic estradiol (E2) 11β-derivatives, 11β-bromoacetamidoethoxyphenylE2 (11BAEOPE2) and 11β-bromoacetamidopentoxyphenylE2 (11BAPOPE2) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERα. To identify and quantify the electrophile Covalent Attachment sites on LBD, [14C]11BAEOPE2- and [14C]11BAPOPE2-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE2 and 11BAPOPE2, respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys530Lys531 LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the Covalent Attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD–ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys530 could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common Attachment to Cys530 of estrogenic E2 17α-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor α, J. Steroid Biochem. Mol. Biol. 98 (4–5), in press] and antiestrogenic E2 11β-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.
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Identification of Covalent Attachment site of antiestrogenic estradiol 11 beta-derivatives on human estrogen receptor alpha ligand-binding domain.
The Journal of steroid biochemistry and molecular biology, 2006Co-Authors: Sigrid Aliau, Hélène Mattras, Jean-louis BorgnaAbstract:Affinity labeling of human estrogen receptor alpha (ERalpha) by high affinity and antiestrogenic estradiol (E(2)) 11 beta-derivatives, 11 beta-bromoacetamidoethoxyphenylE(2) (11BAEOPE(2)) and 11 beta-bromoacetamidopentoxyphenylE(2) (11BAPOPE(2)) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERalpha. To identify and quantify the electrophile Covalent Attachment sites on LBD, [(14)C]11BAEOPE(2)- and [(14)C]11BAPOPE(2)-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE(2) and 11BAPOPE(2), respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys(530)Lys(531) LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the Covalent Attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD-ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys(530) could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common Attachment to Cys(530) of estrogenic E(2) 17 alpha-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor alpha, J. Steroid Biochem. Mol. Biol. 98 (4-5), in press] and antiestrogenic E(2) 11 beta-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.
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Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol Covalent Attachment sites on estrogen receptor alpha.
Biochemistry, 2002Co-Authors: Hélène Mattras, Sigrid Aliau, Eric Richard, Jean-claude Bonnafous, Patrick Jouin, Jean-louis BorgnaAbstract:Mass spectrometry was used to identify the sites of Covalent Attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). A glutathione S-transferase (GST)-LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) Covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 25%) were detected by SDS-PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): two main signals were consistent with the mass of the full-length (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and Covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the Covalent Attachment sites of 17BAPE(2). HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative Covalent Attachment sites of 17BAPE(2) on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol-LBD and raloxifene- or 4-hydroxytamoxifen-LBD complexes. The Covalent Attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD-17BAPE(2) complex.
Hélène Mattras - One of the best experts on this subject based on the ideXlab platform.
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Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor α
The Journal of Steroid Biochemistry and Molecular Biology, 2006Co-Authors: Hélène Mattras, Sigrid Aliau, Emmanuelle Demey, Joël Poncet, Jean-louis BorgnaAbstract:Abstract A purified preparation of human estrogen receptor α (hERα) ligand-binding domain (LBD) involving mainly the Ser309Ala569 (∼30%) and Ser309Ala571 (∼63%) ER portions was used to identify the Covalent Attachment sites of two closely related estrogenic ER affinity labels 17α-bromoacetamidopropylestradiol (17BAPE2) and 17α-bromoacetamidomethylestradiol (17BAME2). To identify and quantify the electrophile Covalent Attachment sites, [14C]17BAPE2- and [14C]17BAME2-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys530Lys531 and Cys417Arg434 hLBD tryptic peptides. The Covalent Attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys530 and Cys417. However, the balance between Cys530 and Cys417 labeling markedly differed according to the affinity label used, with the Cys530/Cys417 ratio being 2.1 for 17BAPE2, and 20 for 17BAME2. We attempted to interpret the Covalent Attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E2. In agreement with the different levels of Cys417 alkylation, the LBD model with unchanged helices could not easily account for Cys417 labeling by 17BAME2, whereas favorable results were obtained through 17BAPE2 docking. Moreover, labeling at Cys530 by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E2 17α-derivatives differ from the structure of crystallized LBD bound to E2.
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Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor alpha.
The Journal of steroid biochemistry and molecular biology, 2006Co-Authors: Hélène Mattras, Sigrid Aliau, Emmanuelle Demey, Joël Poncet, Jean-louis BorgnaAbstract:A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the Covalent Attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile Covalent Attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The Covalent Attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the Covalent Attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).
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Identification of Covalent Attachment site of antiestrogenic estradiol 11β-derivatives on human estrogen receptor α ligand-binding domain
The Journal of Steroid Biochemistry and Molecular Biology, 2006Co-Authors: Sigrid Aliau, Hélène Mattras, Jean-louis BorgnaAbstract:Abstract Affinity labeling of human estrogen receptor α (ERα) by high affinity and antiestrogenic estradiol (E2) 11β-derivatives, 11β-bromoacetamidoethoxyphenylE2 (11BAEOPE2) and 11β-bromoacetamidopentoxyphenylE2 (11BAPOPE2) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERα. To identify and quantify the electrophile Covalent Attachment sites on LBD, [14C]11BAEOPE2- and [14C]11BAPOPE2-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE2 and 11BAPOPE2, respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys530Lys531 LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the Covalent Attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD–ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys530 could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common Attachment to Cys530 of estrogenic E2 17α-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor α, J. Steroid Biochem. Mol. Biol. 98 (4–5), in press] and antiestrogenic E2 11β-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.
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Identification of Covalent Attachment site of antiestrogenic estradiol 11 beta-derivatives on human estrogen receptor alpha ligand-binding domain.
The Journal of steroid biochemistry and molecular biology, 2006Co-Authors: Sigrid Aliau, Hélène Mattras, Jean-louis BorgnaAbstract:Affinity labeling of human estrogen receptor alpha (ERalpha) by high affinity and antiestrogenic estradiol (E(2)) 11 beta-derivatives, 11 beta-bromoacetamidoethoxyphenylE(2) (11BAEOPE(2)) and 11 beta-bromoacetamidopentoxyphenylE(2) (11BAPOPE(2)) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERalpha. To identify and quantify the electrophile Covalent Attachment sites on LBD, [(14)C]11BAEOPE(2)- and [(14)C]11BAPOPE(2)-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE(2) and 11BAPOPE(2), respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys(530)Lys(531) LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the Covalent Attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD-ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys(530) could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common Attachment to Cys(530) of estrogenic E(2) 17 alpha-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor alpha, J. Steroid Biochem. Mol. Biol. 98 (4-5), in press] and antiestrogenic E(2) 11 beta-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.
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Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol Covalent Attachment sites on estrogen receptor alpha.
Biochemistry, 2002Co-Authors: Hélène Mattras, Sigrid Aliau, Eric Richard, Jean-claude Bonnafous, Patrick Jouin, Jean-louis BorgnaAbstract:Mass spectrometry was used to identify the sites of Covalent Attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). A glutathione S-transferase (GST)-LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) Covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 25%) were detected by SDS-PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): two main signals were consistent with the mass of the full-length (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and Covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the Covalent Attachment sites of 17BAPE(2). HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative Covalent Attachment sites of 17BAPE(2) on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol-LBD and raloxifene- or 4-hydroxytamoxifen-LBD complexes. The Covalent Attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD-17BAPE(2) complex.
Jose M. Guisan - One of the best experts on this subject based on the ideXlab platform.
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Immobilization and stabilization of commercial β-1,4-endoxylanase Depol™ 333MDP by multipoint Covalent Attachment for xylan hydrolysis: Production of prebiotics (xylo-oligosaccharides)
Biocatalysis and Biotransformation, 2017Co-Authors: Sandro Martins De Oliveira, Gloria Fernández-lorente, Sonia Moreno-pérez, Maria Romero-fernández, Javier Rocha-martín, Jose M. GuisanAbstract:The commercial enzyme Depol™ 333MDP (D333MDP) was immobilized by multipoint Covalent Attachment onto 10% cross-linked agarose beads support highly activated with aldehyde groups. The enzyme immobil...
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Immobilization of Yarrowia lipolytica lipase--a comparison of stability of physical adsorption and Covalent Attachment techniques
Applied Biochemistry and Biotechnology, 2007Co-Authors: Aline G. Cunha, Gloria Fernández-lorente, Juliana V. Bevilaqua, Jacqueline Destain, Lucia M.c. Paiva, Denise M.g. Freire, Roberto Fernandez-lafuente, Jose M. GuisanAbstract:Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and Covalent Attachment. The enzyme was adsorbed on octyl–agarose and octadecyl–sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE–agarose support by ionic adsorption. CNBr–agarose was used as support for the Covalent Attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl–agarose, octadecyl–sepabeads and MANAE–agarose, respectively. However, the activity retention was lower (34% for octyl–agarose, 50% for octadecyl–sepabeads and 61% for MANAE–agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by Covalent Attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl–sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl–sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl–agarose and MANAE–agarose supports presented low stability, even less than the free enzyme.
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Immobilization of Yarrowia lipolytica lipase--a comparison of stability of physical adsorption and Covalent Attachment techniques.
Applied biochemistry and biotechnology, 2007Co-Authors: Aline G. Cunha, Gloria Fernández-lorente, Juliana V. Bevilaqua, Jacqueline Destain, Lucia M.c. Paiva, Denise M.g. Freire, Roberto Fernandez-lafuente, Jose M. GuisanAbstract:Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and Covalent Attachment. The enzyme was adsorbed on octyl-agarose and octadecyl-sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE-agarose support by ionic adsorption. CNBr-agarose was used as support for the Covalent Attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl-agarose, octadecyl-sepabeads and MANAE-agarose, respectively. However, the activity retention was lower (34% for octyl-agarose, 50% for octadecyl-sepabeads and 61% for MANAE-agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by Covalent Attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45 degrees C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl-sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl-sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl-agarose and MANAE-agarose supports presented low stability, even less than the free enzyme.
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Improved stabilization of chemically aminated enzymes via multipoint Covalent Attachment on glyoxyl supports
Journal of biotechnology, 2004Co-Authors: Fernando López-gallego, Jose M. Guisan, Tamara Montes, Manuel Fuentes, Noelia Alonso, Valeria Grazú, Lorena Betancor, Roberto Fernandez-lafuenteAbstract:The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint Covalent Attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pKa of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.
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Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint Covalent Attachment☆
Enzyme and microbial technology, 2000Co-Authors: Cesar Mateo, Olga Abian, Roberto Fernandez Lafuente, Jose M. GuisanAbstract:Abstract Epoxy supports (Eupergit C) may be very suitable to achieve the multipoint Covalent Attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. To achieve a significant multipoint Covalent Attachment, the control of the experimental conditions was found to be critical. A three-step immobilization/stabilization procedure is here proposed: 1) the enzyme is firstly Covalently immobilized under very mild experimental conditions (e.g. pH 7.0 and 20°C); 2) the already immobilized enzyme is further incubated under more drastic conditions (higher pH values, longer incubation periods, etc.) to “facilitate” the formation of new Covalent linkages between the immobilized enzyme molecule and the support; 3) the remaining groups of the support are blocked to stop any additional interaction between the enzyme and the support. Progressive establishment of new enzyme-support Attachments was showed by the progressive irreversible Covalent immobilization of several subunits of multi-subunits proteins (all non-Covalent structures contained in crude extracts of different microorganism, penicillin G acylase and chymotrypsin). This multipoint Covalent Attachment enabled the significant thermostabilization of two relevant enzymes, (compared with the just immobilized derivatives): chymotrypsin (5-fold factor) and penicillin G acylase (18-fold factor). Bearing in mind that this stabilization was additive to that achieved by conventional immobilization, the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative. These stabilizations were observed also when the inactivations were promoted by the enzyme exposure to drastic pH values or the presence of cosolvents.
Sigrid Aliau - One of the best experts on this subject based on the ideXlab platform.
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Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor α
The Journal of Steroid Biochemistry and Molecular Biology, 2006Co-Authors: Hélène Mattras, Sigrid Aliau, Emmanuelle Demey, Joël Poncet, Jean-louis BorgnaAbstract:Abstract A purified preparation of human estrogen receptor α (hERα) ligand-binding domain (LBD) involving mainly the Ser309Ala569 (∼30%) and Ser309Ala571 (∼63%) ER portions was used to identify the Covalent Attachment sites of two closely related estrogenic ER affinity labels 17α-bromoacetamidopropylestradiol (17BAPE2) and 17α-bromoacetamidomethylestradiol (17BAME2). To identify and quantify the electrophile Covalent Attachment sites, [14C]17BAPE2- and [14C]17BAME2-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys530Lys531 and Cys417Arg434 hLBD tryptic peptides. The Covalent Attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys530 and Cys417. However, the balance between Cys530 and Cys417 labeling markedly differed according to the affinity label used, with the Cys530/Cys417 ratio being 2.1 for 17BAPE2, and 20 for 17BAME2. We attempted to interpret the Covalent Attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E2. In agreement with the different levels of Cys417 alkylation, the LBD model with unchanged helices could not easily account for Cys417 labeling by 17BAME2, whereas favorable results were obtained through 17BAPE2 docking. Moreover, labeling at Cys530 by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E2 17α-derivatives differ from the structure of crystallized LBD bound to E2.
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Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor alpha.
The Journal of steroid biochemistry and molecular biology, 2006Co-Authors: Hélène Mattras, Sigrid Aliau, Emmanuelle Demey, Joël Poncet, Jean-louis BorgnaAbstract:A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the Covalent Attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile Covalent Attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The Covalent Attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the Covalent Attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).
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Identification of Covalent Attachment site of antiestrogenic estradiol 11β-derivatives on human estrogen receptor α ligand-binding domain
The Journal of Steroid Biochemistry and Molecular Biology, 2006Co-Authors: Sigrid Aliau, Hélène Mattras, Jean-louis BorgnaAbstract:Abstract Affinity labeling of human estrogen receptor α (ERα) by high affinity and antiestrogenic estradiol (E2) 11β-derivatives, 11β-bromoacetamidoethoxyphenylE2 (11BAEOPE2) and 11β-bromoacetamidopentoxyphenylE2 (11BAPOPE2) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERα. To identify and quantify the electrophile Covalent Attachment sites on LBD, [14C]11BAEOPE2- and [14C]11BAPOPE2-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE2 and 11BAPOPE2, respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys530Lys531 LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the Covalent Attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD–ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys530 could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common Attachment to Cys530 of estrogenic E2 17α-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor α, J. Steroid Biochem. Mol. Biol. 98 (4–5), in press] and antiestrogenic E2 11β-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.
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Identification of Covalent Attachment site of antiestrogenic estradiol 11 beta-derivatives on human estrogen receptor alpha ligand-binding domain.
The Journal of steroid biochemistry and molecular biology, 2006Co-Authors: Sigrid Aliau, Hélène Mattras, Jean-louis BorgnaAbstract:Affinity labeling of human estrogen receptor alpha (ERalpha) by high affinity and antiestrogenic estradiol (E(2)) 11 beta-derivatives, 11 beta-bromoacetamidoethoxyphenylE(2) (11BAEOPE(2)) and 11 beta-bromoacetamidopentoxyphenylE(2) (11BAPOPE(2)) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERalpha. To identify and quantify the electrophile Covalent Attachment sites on LBD, [(14)C]11BAEOPE(2)- and [(14)C]11BAPOPE(2)-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE(2) and 11BAPOPE(2), respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys(530)Lys(531) LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the Covalent Attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD-ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys(530) could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common Attachment to Cys(530) of estrogenic E(2) 17 alpha-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of Covalent Attachment sites of two related estrogenic ligands on human estrogen receptor alpha, J. Steroid Biochem. Mol. Biol. 98 (4-5), in press] and antiestrogenic E(2) 11 beta-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.
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Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol Covalent Attachment sites on estrogen receptor alpha.
Biochemistry, 2002Co-Authors: Hélène Mattras, Sigrid Aliau, Eric Richard, Jean-claude Bonnafous, Patrick Jouin, Jean-louis BorgnaAbstract:Mass spectrometry was used to identify the sites of Covalent Attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). A glutathione S-transferase (GST)-LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) Covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 25%) were detected by SDS-PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): two main signals were consistent with the mass of the full-length (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and Covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the Covalent Attachment sites of 17BAPE(2). HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative Covalent Attachment sites of 17BAPE(2) on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol-LBD and raloxifene- or 4-hydroxytamoxifen-LBD complexes. The Covalent Attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD-17BAPE(2) complex.
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Non-Covalent Attachment of proteins to single-walled carbon nanotubes.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Luis F F Neves, Ta Wei Tsai, Naveen R. Palwai, David E. Martyn, Yongqiang Tan, David W. Schmidtke, Daniel E. Resasco, Roger G. HarrisonAbstract:A method for the non-Covalent Attachment of proteins to single-walled carbon nanotubes (SWNTs) is described. In this method, the protein is adsorbed to SWNTs that are suspended using sodium cholate, a surfactant and bile salt. The sodium cholate is then removed by dialysis with retention of the protein on the SWNTs. This method has resulted in good protein loadings and good retention of protein activity.
