Research and training in structural genomics and molecular biophysics for the characterization and design of proteins and enzymes

Abstract

To perform their biological functions, proteins must fold into defined three-dimensional structures. These structures are highly conserved, such that ~92 million protein sequences are classifiable in only ~6,000 structures. In these scaffolds, residues that are essential for the chemical origin of protein function are placed in specific positions, thus being highly conserved between protein sequences that perform similar functions. On the contrary, other residues within these functional sites show variations between protein sequences and are largely responsible to encode differences in specificity for a particular binding partner. In this scenario, the deluge of protein sequences derived from genomic data during the last decades has been fundamental for the identification of mutations responsible for loss of protein function, as well as for the discovery of enzymes that catalyze novel reactions in nature. However, sequence variation and missanotation of protein sequences using bioinformatics limits their finding. Thus, researchers opted for accompanying genomic analysis with biophysical, biochemical and structural characterizations of proteins. Our proposal aims to combine these disciplines into a Structural Genomics and Molecular Biophysics scheme. The main goal is to train both students and researchers into the proper combination and use of computational and experimental approaches from each discipline, using a research-based approach based on the exemplar case of enzymes that degrade the synthetic plastic PET, which is an environmentally threatening waste constantly being accumulated in landfills and oceans at rates paralleling their production. These enzymes have a highly conserved structure and catalytic residues, but their molecular mechanism and the sequence-structure variations enabling the activity of some of these enzymes at room temperature is poorly understood. In this context, we will combine the efforts from research teams at P. Universidad Católica de Chile and Universidade de São Paulo in the identification of PET-degrading enzymes from Antarctic metagenomes, computational prediction of their PET-degrading potential, biochemical characterization of their activity and thermal stability, and determination of their protein structure to identify the variations encoding these activity-stability traits to foster protein engineering of these enzymes. The development of this project will enable the training of students in metagenomics, biophysics, biochemistry and structural biology from both research teams, and to disseminate our work and findings among our scientific communities. (AU)