STRUCTURAL STABILIZATION OF AN OLIGOMERIC SCAFFOLD AT ACIDIC PH FOR THE PRODUCTION OF ARTIFICIAL CELLULOSOMES

GARRONE NA.; GOLDBAUM FA.; CRAIG PO.

CABA

Jornada; Jornadas Interdisciplinarias de Química Biológica; 2017

Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires

The polymeric display of molecules is an emerging technology for many applications. It could be used to increase the immunogenicity of antigens, the interaction strength of protein domains for polymeric ligands, and the production of multifunctional particles. We use a oligomeric protein scaffold for the polymeric display of protein targets. Coupling to the scaffold is produced through a non-covalent strategy with high affinity heterodimeric modules complementary fused to the scaffold and the target proteins. We are using this method for the development of artificial cellulosomes based on the colocalization of lignocellulolytic enzymes and cellulose binding domains. This colocalización would increase cellulose degradation activity through enzyme proximity and targeting effects and would be valuable for the production of biofuels. However, the decameric structure of the scaffold dissociates into pentamers below pH 6, a condition in whichcertain cellulolytic enzymes exhibit maximum activity. The objective of this work is the stabilization of the pentamer-pentamer interface of the scaffold by means of protein engineering to improve its properties as a structural scaffold of artificial cellulosomes at acidic pH. The dissociation of pentamers at acidic pH seems to be induced by protonation of histidines at the pentamer-pentamer interface, which would decrease the level of colocalization of the enzymes coupled to the scaffold. To design stabilizing mutations, we bioinformatically explored with FOLDX software all possible single mutants of the scaffold and their effect on its stability at pH 5 and pH 7. From this analysis we selected those single mutants which showed a significant stabilization at the interface at pH 5, no destabilization at pH 7, and no destabilization of the pentamer structure at pH5 or pH7. The combination of the selected mutations and their effect on the stability of the scaffold as a function of pH is also presented.