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Fluorescence of the Single Tryptophan of Cutinase: Temperature and pH Effect on Protein Conformation and Dynamics ¶
Author(s) -
Martinho J. M. G.,
Santos A. M.,
Fedorov A.,
Baptista R. P.,
Taipa M. A.,
Cabral J. M. S.
Publication year - 2003
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/0031-8655(2003)0780015fotsto2.0.co2
Subject(s) - chemistry , tryptophan , fluorescence , cysteine , cystine , residue (chemistry) , cutinase , denaturation (fissile materials) , crystallography , photochemistry , stereochemistry , enzyme , biochemistry , amino acid , physics , quantum mechanics , nuclear chemistry
The cutinase from Fusarium solani pisi is an enzyme with a single l ‐tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala–Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge–transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10‐fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling‐in‐cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein‐melting temperature and to protein‐backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.