Orientation of Cutinase Adsorbed onto PMMA Nanoparticles Probed by Tryptophan Fluorescence
The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d = 106.0 ± 0.1 nm) were coated wit...
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Veröffentlicht in: | The journal of physical chemistry. B 2008-03, Vol.112 (12), p.3581-3585 |
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creator | Santos, Andrea M Fedorov, Aleksander Martinho, José M. G Baptista, Ricardo P Taipa, Maria Ângela Cabral, Joaquim M. S |
description | The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d = 106.0 ± 0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3−0.4, 2−3, and 6−7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp−Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH. |
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G ; Baptista, Ricardo P ; Taipa, Maria Ângela ; Cabral, Joaquim M. S</creator><creatorcontrib>Santos, Andrea M ; Fedorov, Aleksander ; Martinho, José M. G ; Baptista, Ricardo P ; Taipa, Maria Ângela ; Cabral, Joaquim M. S</creatorcontrib><description>The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d = 106.0 ± 0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3−0.4, 2−3, and 6−7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp−Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH.</description><identifier>ISSN: 1520-6106</identifier><identifier>EISSN: 1520-5207</identifier><identifier>DOI: 10.1021/jp711903e</identifier><identifier>PMID: 18311968</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Absorption ; Carboxylic Ester Hydrolases - chemistry ; Disulfides - chemistry ; Fluorescence ; Fusarium - enzymology ; Models, Molecular ; Nanoparticles - chemistry ; Polymethyl Methacrylate - chemistry ; Protein Folding ; Protein Structure, Tertiary ; Tryptophan - analysis ; Tryptophan - chemistry</subject><ispartof>The journal of physical chemistry. 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G</creatorcontrib><creatorcontrib>Baptista, Ricardo P</creatorcontrib><creatorcontrib>Taipa, Maria Ângela</creatorcontrib><creatorcontrib>Cabral, Joaquim M. S</creatorcontrib><title>Orientation of Cutinase Adsorbed onto PMMA Nanoparticles Probed by Tryptophan Fluorescence</title><title>The journal of physical chemistry. B</title><addtitle>J. Phys. Chem. B</addtitle><description>The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d = 106.0 ± 0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3−0.4, 2−3, and 6−7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp−Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH.</description><subject>Absorption</subject><subject>Carboxylic Ester Hydrolases - chemistry</subject><subject>Disulfides - chemistry</subject><subject>Fluorescence</subject><subject>Fusarium - enzymology</subject><subject>Models, Molecular</subject><subject>Nanoparticles - chemistry</subject><subject>Polymethyl Methacrylate - chemistry</subject><subject>Protein Folding</subject><subject>Protein Structure, Tertiary</subject><subject>Tryptophan - analysis</subject><subject>Tryptophan - chemistry</subject><issn>1520-6106</issn><issn>1520-5207</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNptkEtLxDAQx4Movg9-AclFwUM1aZs0PS6LT3ysuIp4CWk6xa7dpCYpuN_eyC568TDMMPOb1x-hA0pOKUnp2awvKC1JBmtom7KUJNGK9VXMKeFbaMf7GSEpSwXfRFtUZLGBi2309uBaMEGF1hpsGzweQmuUBzyqvXUV1NiaYPHk7m6E75WxvXKh1R14PHH2p1wt8NQt-mD7d2XwRTdYB16D0bCHNhrVedhf-V30fHE-HV8ltw-X1-PRbaIyRkMiyrRuRK455FozkUGmeS2quhBlCYxxWgtoqqqMyfhVlQsBnNcVYVQBi73ZLjpezu2d_RzABzlv4wVdpwzYwcuC5DSjpYjgyRLUznrvoJG9a-fKLSQl8kdI-StkZA9XQ4dqDvUfuVIuAskSaH2Ar9-6ch-SF1nB5HTyJMdvN_nL6-OVfIr80ZJX2suZHZyJmvyz-Bsnhonc</recordid><startdate>20080327</startdate><enddate>20080327</enddate><creator>Santos, Andrea M</creator><creator>Fedorov, Aleksander</creator><creator>Martinho, José M. 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S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a351t-892df84c6e4cc583e3c6d8bd7899e5561d8efbb96d8207b488e66db051ae592d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Absorption</topic><topic>Carboxylic Ester Hydrolases - chemistry</topic><topic>Disulfides - chemistry</topic><topic>Fluorescence</topic><topic>Fusarium - enzymology</topic><topic>Models, Molecular</topic><topic>Nanoparticles - chemistry</topic><topic>Polymethyl Methacrylate - chemistry</topic><topic>Protein Folding</topic><topic>Protein Structure, Tertiary</topic><topic>Tryptophan - analysis</topic><topic>Tryptophan - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Santos, Andrea M</creatorcontrib><creatorcontrib>Fedorov, Aleksander</creatorcontrib><creatorcontrib>Martinho, José M. 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S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Orientation of Cutinase Adsorbed onto PMMA Nanoparticles Probed by Tryptophan Fluorescence</atitle><jtitle>The journal of physical chemistry. B</jtitle><addtitle>J. Phys. Chem. B</addtitle><date>2008-03-27</date><risdate>2008</risdate><volume>112</volume><issue>12</issue><spage>3581</spage><epage>3585</epage><pages>3581-3585</pages><issn>1520-6106</issn><eissn>1520-5207</eissn><abstract>The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d = 106.0 ± 0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3−0.4, 2−3, and 6−7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp−Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>18311968</pmid><doi>10.1021/jp711903e</doi><tpages>5</tpages></addata></record> |
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subjects | Absorption Carboxylic Ester Hydrolases - chemistry Disulfides - chemistry Fluorescence Fusarium - enzymology Models, Molecular Nanoparticles - chemistry Polymethyl Methacrylate - chemistry Protein Folding Protein Structure, Tertiary Tryptophan - analysis Tryptophan - chemistry |
title | Orientation of Cutinase Adsorbed onto PMMA Nanoparticles Probed by Tryptophan Fluorescence |
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