De novo design, synthesis and screening of a combinatorial library of complementary ligands directed towards the surface of cutinase from Fusarium solani pisi

Abstract

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.

Synthetic affinity ligands as a novel tool to improve protein stability

“…The first library screened to search for affinity ligands with a potential stabilizing effect on cutinase (Figure 2) resulted from a rational approach, based on molecular modelling tools, to design affinity ligands with the ability to bind cutinase in identified unfolding regions (Ruiu et al, 2006). The most efficient ligand design approaches are, in general, those based on mimicking natural occurring interactions in biological complexes (templates) such as enzyme/substrate or enzyme/coenzyme, for example.…”

“…The libraries of ligands used in the present work were synthesized in solid-phase by a well-established 'mix-and-split' combinatorial method as described elsewhere (Roque et al, 2005;Ruiu et al, 2006). All ligands used a 1,3,5-trichloro-sym-triazine (cyanuric chloride) scaffold as a spatial framework displaying two different substituent groups.…”

“…Selected ligands from the random combinatorial library were assessed for binding cutinase by affinity chromatography as previously described (Ruiu et al, 2006). An amount of 0.5 ml of each affinity matrix was packed into a small column and washed with regeneration solution (0.1 M NaOH in 30% (v/v) isopropanol) and with distilled water to bring the pH to neutral.…”

“…This knowledge can provide guidelines for different types of protein stabilization strategies (e.g. generation of improved variants of cutinase by means of protein engineering) and was recently used as the basis for the rational design of a solid-phase combinatorial library of triazine-based affinity ligands directed towards the surface of cutinase (Ruiu et al, 2006). The rationale of this work is that if such ligands are able to specifically bind by affinity-like interactions to these flexible unfolding regions, they are likely to improve protein stability.…”

“…Substituents that gave consistently good results were combined with others previously selected (Ruiu et al, 2006) from a rationally designed library, and used to synthesize a second-generation biased library, following a semi-rational approach. Ligands efficiently binding to cutinase while maintaining high biological activity were utilized to perform thermostability assays with adsorbed/immobilized cutinase.…”