Local environment perturbations in alpha1-antitrypsin monitored by a ratiometric fluorescent label.
The complex multistep inhibition of proteinases by alpha(1)-antitrypsin (alpha(1)-AT) was investigated by covalently labeling its unique Cys residue with a ratiometric environment-sensitive fluorescent dye, 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone (BMFC). The binding of BMFC-labeled alpha(1)-AT with pancreatic elastase led to significant changes in the dual emission of BMFC. The 8 nm blue shift of one of the bands and ca. 65% change in the intensity ratio of the two emission bands suggested an increased exposure of the labeled Cys-232 residue to the bulk water on complex formation. In contrast, the bacterial V8 proteinase-induced cleavage of the reactive center loop of BMFC-labeled alpha(1)-AT did not generate any significant change in the Cys-232 region. Similar experiments with elastase and alpha(1)-AT conjugated to the classical environment-sensitive dye, IANBD, confirmed these results but led to much smaller modifications in the emission spectrum. Stopped-flow investigation of the reaction between BMFC-labeled alpha(1)-AT and elastase showed both a well-described fast and a new slow step of the inhibition process. The latter step is probably associated with the structural reorganization aimed at stabilizing the final complex. These results present a convenient fluorescence ratiometric approach based on the BMFC label for studies of protein conformational changes.
Boudier
Christian
C
Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Faculté de Pharmacie, Université de Strasbourg, 67401 Illkirch-Cedex, France.
Klymchenko
Andrey S
AS
Mely
Yves
Y
Follenius-Wund
Anny
A
D000818:Animals; D002867:Chromones / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010069:Oxadiazoles / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013211:Staphylococcus aureus / Q000201:enzymology; D013552:Swine; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
C506578:6-bromomethyl-2-(2-furanyl)-3-hydroxychromone; D002867:Chromones; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D000515:alpha 1-Antitrypsin; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase; D003545:Cysteine