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
Hauptverfasser: Santos, Andrea M, Fedorov, Aleksander, Martinho, José M. G, Baptista, Ricardo P, Taipa, Maria Ângela, Cabral, Joaquim M. S
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container_end_page 3585
container_issue 12
container_start_page 3581
container_title The journal of physical chemistry. B
container_volume 112
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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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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