Partial identification of carbohydrate-binding sites of a Galα1,3Galβ1,4GlcNAc-specific lectin from the mushroom Marasmius oreades by site-directed mutagenesis

2004 ◽  
Vol 427 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Hiroaki Tateno ◽  
Irwin J Goldstein
Keyword(s):  
Binding Sites ◽  
Partial Identification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbohydrate Binding

scholarly journals Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa

Glycobiology ◽  
2020 ◽  
Author(s):  
Elena Shanina ◽  
Eike Siebs ◽  
Hengxi Zhang ◽  
Daniel Varón Silva ◽  
Ines Joachim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbohydrate Binding ◽  
Binding Region ◽  
Chronic Infections ◽  
Nmr Titration ◽  
Affinity Ligands ◽  
Natural Ligands ◽  
Future Drug

Abstract The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis

2009 ◽  
Vol 1790 (10) ◽  
pp. 1327-1333 ◽  
Author(s):  
Tatsuya Ueki ◽  
Norifumi Kawakami ◽  
Masaaki Toshishige ◽  
Koichi Matsuo ◽  
Kunihiko Gekko ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites

Biochemistry ◽  
1992 ◽  
Vol 31 (24) ◽  
pp. 5449-5458 ◽  
Author(s):  
John Jenkins ◽  
Joel Janin ◽  
Felix Rey ◽  
Mohammed Chiadmi ◽  
Herman Van Tilbeurgh ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Metal Binding ◽  
Binding Sites ◽  
Glucose Isomerase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Metal Binding Sites
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