title: Protein stabilization with a dipeptide-mimic triazine-scaffolded synthetic affinity ligand.
abstract: Protein stabilization was achieved by a novel approach based on the adsorption and establishment of affinity-like interactions with a biomimetic triazine-scaffolded ligand. A synthetic lead compound (ligand 3'/11, K(a) ≈ 10(4) M(-1)) was selected from a previously screened solid-phase library of affinity ligands for studies of adsorption and stabilization of cutinase from Fusarium solani pisi used as a model system. This ligand, directly synthesized in agarose by a well-established solid-phase synthesis method, was able to strongly bind cutinase and led to impressive half-lives of more than 8 h at 70 °C, and of approximately 34 h at 60 °C for bound protein (a 25- and 57-fold increase as compared with the free enzyme, respectively). The ligand density in the solid matrix was found to be a determinant parameter for cutinase stabilization. It is conceivable that the highly stabilizing effect observed results from the binding of more than one ligand residue to the enzyme, creating specific macromolecular configurations that lock structural mobility thus improving molecular stability.
authors: Institute for Biotechnology and Bioengineering, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av Rovisco Pais, 1049-001 Lisboa, Portugal.
authors: I T
authors: IT
authors: Sousa
authors: N M T
authors: NM
authors: Lourenço
authors: C A M
authors: CA
authors: Afonso
authors: M A
authors: MA
authors: Taipa
mesh_terms: D000327:Adsorption; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D004151:Dipeptides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005079:Excipients / Q000138:chemical synthesis* / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005670:Fusarium; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D008024:Ligands; D018716:Molecular Mimicry; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012685:Sepharose / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques; D013816:Thermodynamics; D014227:Triazines / Q000138:chemical synthesis* / Q000737:chemistry; D062366:para-Aminobenzoates / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D004151:Dipeptides; D005079:Excipients; D005656:Fungal Proteins; D008024:Ligands; D011994:Recombinant Proteins; D014227:Triazines; D062366:para-Aminobenzoates; D012685:Sepharose; D002265:Carboxylic Ester Hydrolases; C019952:cutinase