De novo design, synthesis and screening of a combinatorial library of complementary ligands directed towards the surface of cutinase from Fusarium solani pisi.
The protein surface is the interface through which a protein molecule senses the external world. The composition of this interface, in charged, polar and/or hydrophobic residues is crucial for both the activity and stability of the protein. Protein immobilization on surfaces has been extensively explored as one of the most effective approaches for stabilization. The mechanism of stabilization, however, is still poorly understood, and usually the success of any method is more a matter of trial and error rather than the result of rational concepts. The importance of local unfolding processes in a number of biologically significant processes has been recognized and attracted increasing attention. Unfolding regions have been localized in different proteins including the recombinant cutinase from Fusarium solani pisi. The study of three structural surface regions associated with early cutinase unfolding events was the basis for the approach followed in this work. A 64-member solid-phase combinatorial library of ligands was synthesized on a triazine-substituted agarose matrix using a modified 'mix and split' procedure. The combinatorial library was assessed for binding to cutinase from Fusarium solani pisi in a biologically active form. Four lead ligands (3/5, 3/7, 4/5, 4/7) have been selected in which immobilized cutinase presented a relative activity of 30-60% as compared to the free enzyme.
Ruiu
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Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal.
Roque
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AC
Taipa
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MA
Lowe
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CR
D000327:Adsorption; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D018360:Crystallography, X-Ray; D030562:Databases, Protein; D004800:Enzymes, Immobilized / Q000737:chemistry; D005670:Fusarium / Q000201:enzymology*; D008024:Ligands; D008958:Models, Molecular; D019151:Peptide Library
D004800:Enzymes, Immobilized; D008024:Ligands; D019151:Peptide Library; D002265:Carboxylic Ester Hydrolases; C019952:cutinase