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Abstract

Nature has the ability to cope with extreme pH, temperature and pressure, in addition to bestowing a wide array of functionalities to (bio)macromolecules. These specialized skills have attracted the interest of vast scientific communities especially in view of possible industrial or biomedical applications. In general, the design of functional and thermostable biological constructs is one of the overarching themes in biochemisty. Despite extensive research and some recent breakthroughs, the level of understanding of the factors that determine protein reactivity and stability is still modest. The use of computational methods for an atomic level understanding of the functionality and stability of biomacromolecules is therefore an attractive alternative. This dissertation presents a number of research projects that combine different computational methods in a close blend with experiments in order to identify the crucial determinants that can be used for a rational design of thermostable and catalytically active, (re-)engineered protein domains. The main findings are the following: Chapter 3 of the thesis presents the results of computational studies towards the design of biomimetic compounds capable of performing tetrazole formation based on Lewis acid catalysis. Our quantum chemical calculations suggest the incorporation of unnatural modifications of glutamate residues as a promising starting point for the development of an efficacious bioinspired catalytic system based on a zinc active site. Chapter 4 and 5 focuses on the computational design and re-engineering of the B1 domain of Streptococcal protein G (GB1) to introduce hydrolytic activity. In addition to exhibiting encouraging reaction rates, our computationally designed artificial enzymes are reasonably thermostable. Chapter 6 is about the design of a highly thermostable metallo variant of GB1. It describes the successful attempt to establish a stable tetrahedral zinc binding site in GB1 with the help of a computational protocol based on genetic algorithm optimization. A high resolution crystal structure of this metallo variant reveals a novel head-to-tail organization. Chapter 7 reports the design of two re-engineered thermostable GB1 sequences using a new computational method for thermostability prediction as well as on their expression and structural characterization. Interestingly, one of the mutants show an intertwined dimer organization in the crystal structure. The combined theoretical and experimental approaches reported herein provide valuable insights into the successful design of different GB1 variants with tailored properties and may help to open new avenues for the rational design of new thermostable and functional biomacromolecules.

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