Design and characterization of engineered proteins for biotechnological applications

Protein design is nowadays a powerful tool for developing different strategies that can be used to rationally control the stability, structural dynamics, and functional behaviour of proteins of biotechnological interest. Through a biochemical, structural, and computational approach, this work explores the relationship between targeted structural modifications and emerging properties of the systems studied. The sweet protein MNEI, a single-chain monellin, a model protein for sweetness and aggregation studies, was used to understand how topological interventions of circular permutation or point substitutions can alter the balance between stability, swapped dimer formation, and aggregation. The results show that small variations in chain connectivity are enough to change the protein's fate towards ordered fibrils, amorphous aggregates, while changes in the distribution of internal charges can lead to enhanced structural stability accompanied by a drastic reduction in aggregation. Aggregation is therefore a process that can be modulated through targeted design choices. The second part of the thesis is focusing on PIR (Proteins with Internal Repetitions) proteins, in particular on sequence modularity and the role of internal repeats in cell wall anchoring. The analysis of single-repeat constructs made it possible to investigate their contribution to structural stability and interaction with carbohydrates, highlighting a relationship between modular organization and biological function. In summary, the work shows how minimal but strategic changes to the sequence or topology can be exploited to optimize proteins for food, industrial, and biotechnological applications.