Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy.
Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r(0) was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan's presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins.
Department of Biotechnology and Biophysics, Julius-Maximilians University, Wuerzburg, Germany.
Antonia
A
Göhler
Claudia
C
Büchner
Sabine
S
André
Sören
S
Doose
Herbert
H
Kaltner
H-J
HJ
Gabius
D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D005454:Fluorescence Polarization; D037483:Galectin 1 / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D012997:Solvents / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*; D014783:Viscosity
D002241:Carbohydrates; D037483:Galectin 1; D008024:Ligands; D012997:Solvents; D014364:Tryptophan; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases