title: Synthetic affinity ligands as a novel tool to improve protein stability.
abstract: Cutinase from Fusarium solani pisi is the model-system for a new approach to assess and enhance protein stability based on the use of synthetic triazine-scaffolded affinity ligands as a novel protein-stabilizing tool. The active site of cutinase is excluded from the main surface regions postulated to be involved in early protein's thermal unfolding events. Hence, these regions are suitable targets for binding complementary affinity ligands with a potential stabilizing effect. A random solid-phase combinatorial library of triazine-bisubstituted molecules was screened for binding cutinase by a rapid fluorescence-based method and affinity chromatography. The best binding substituents were combined with those previously selected by screening a rationally designed library. A second-generation solid-phase biased library was designed and synthesized, following a semi-rational methodology. A dual screening of this library enabled the selection of ligands binding cutinase with higher affinity while retaining its functionality. These compounds were utilized for thermostability assessment with adsorbed cutinase at 60 degrees C and pH 8.0. When bound to different types of ligands, the enzyme showed markedly distinct activity retention profiles, with some synthetic affinity ligands displaying a stabilizing effect on cutinase and others a clearly destabilizing effect, when compared with the free enzyme.
authors.affiliation: Institute for Biotechnology and Bioengineering (IBB), Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors.forename: I T
authors.initials: IT
authors.lastname: Sousa
authors.forename: L
authors.initials: L
authors.lastname: Ruiu
authors.forename: C R
authors.initials: CR
authors.lastname: Lowe
authors.forename: M A
authors.initials: MA
authors.lastname: Taipa
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D020650:Combinatorial Chemistry Techniques; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005670:Fusarium / Q000201:enzymology / Q000235:genetics; D008024:Ligands; D008958:Models, Molecular; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014227:Triazines / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D004800:Enzymes, Immobilized; D008024:Ligands; D019151:Peptide Library; D011994:Recombinant Proteins; D014227:Triazines; D002265:Carboxylic Ester Hydrolases; C019952:cutinase