Investigation on the inactivation of trypsin by oenothein B: isothermal titration calorimetry and docking studies

2019 ◽  
Vol 55 (3) ◽  
pp. 1351-1358
Author(s):  
Li Wang ◽  
Huijun Liang ◽  
Zitao Liu ◽  
Jingfeng Zhang ◽  
Yu Wang ◽  
...  
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Docking Studies ◽  
Titration Calorimetry ◽  
Oenothein B

scholarly journals A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

2013 ◽  
Vol 289 (3) ◽  
pp. 1377-1387 ◽  
Author(s):  
Jagdeep Kaur ◽  
Elena Olkhova ◽  
Viveka Nand Malviya ◽  
Ernst Grell ◽  
Hartmut Michel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ligand Binding ◽  
Isothermal Titration Calorimetry ◽  
Functional Characterization ◽  
Homology Model ◽  
Docking Studies ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Amino Acid Residues

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

Sign in / Sign up

Export Citation Format

Share Document