Fluorescence of the single tryptophan of cutinase: temperature and pH effect on protein conformation and dynamics.
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.
Martinho
J M G
JM
Centro de Química-Física Molecular, Instituto Superior Técnico, Lisbon, Portugal. jgmartinho@ist.utl.pt
Santos
A M
AM
Fedorov
A
A
Baptista
R P
RP
Taipa
M A
MA
Cabral
J M S
JM
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000528:radiation effects*; D005453:Fluorescence; D005454:Fluorescence Polarization; D005670:Fusarium / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry* / Q000528:radiation effects; D014466:Ultraviolet Rays
D014364:Tryptophan; D002265:Carboxylic Ester Hydrolases; C019952:cutinase