Proteins are shaped by evolution because random mutagenesis, the major force in evolutionary change, leads to random changes in the amino acid sequence. If the activity of the protein is sensitive to the exact nature of many of the amino acid residues, such mutations will often render the protein inactive. Thus, an evolutionary pressure exists to minimize the number of essential amino acid residues in a protein, while maintaining its stability and activity. In this project we distinguish three categories of essential amino acids in enzymes, using β-lactamase as the object of study. First, the residues in the active site that are responsible for substrate binding and catalysis. Second, the surprisingly conserved layer of residues directly surrounding the active site and, third, residues farther from the active site. The focus of this project is on the function of the second and the third group of residues. We propose that the function of the second-shell residues is to position the active site residues to (sub)Ångström precision to allow for catalysis. The third groups comprises residues located on the edges of secondary structure and in coil regions. Within α-helices and β-strands hardly any essential residues are present. We propose a model in which the function of the essential residues is to ‘staple’ the secondary structure into the correct 3D orientation to form the overall frame, bringing the active site residues together. In this model none of residues within the secondary structure elements are essential as long as the secondary structure can be formed. In this way, with few key residues the frame can be produced. The aim of the project is to test these models by a comprehensive mutagenesis study of the group II and III residues of β-lactamase from Mycobacterium tuberculosis. The generated variants will be studied in detail to establish the contributions of residues to the structure, dynamic properties, stability and activity of the enzyme using crystallography, NMR spectroscopy, circular dichroism and kinetic assays. The project will yield an overview of the functions of conserved residues in β-lactamases, as part of a larger line research aimed at better understanding of these enzymes, and ultimately, the design of inhibitors that are less prone to generate resistance. Furthermore, the experiments will test generally applicable models about the roles of essential amino acids outside the enzyme’s active site that can be interpreted in terms of evolutionary protein robustness.

The tertiary structure of proteins is only marginally more stable than the denatured form. This limits the evolution of enzymes to gain new properties, because many random mutations destabilize the tertiary structure. When the destabilization is larger than the buffering capacity of the enzyme stability, the mutated enzyme will lose its structure and thus its function. However, the free energy difference between folded and unfolded protein is temperature dependent, decreasing to zero at the melting temperature. At low temperature an enzyme should, therefore, have a larger buffer against destabilisation than at high temperature. This idea will be tested in the project. The hypothesis is that the temperature at which evolution occurs affects the evolutionary pathway and the conformational landscape of the resulting mutants. We will carry out laboratory evolution experiments using error-prone PCR on the β-lactamase BlaC from Mycobacterium tuberculosis and select for increased turn-over rates of several poor substrates (kcat ~ 1 s-1). Selection will be done at 20, 30 and 37°C and several rounds of directed evolution will be performed. The blaC gene, expressed in E. coli, offers a convenient one-gene/one-phenotype selection system with the antibiotic serving as a strong selection force. The mutants will be characterized for stability, kinetic profile, structure and dynamic properties. Dynamics will be studied at multiple timescales using NMR relaxation techniques. We expect that in particular millisecond dynamics of the new mutants will be dependent on the evolutionary pathway. Using this well-defined system, it can be determined whether the selection at different temperatures leads to different evolutionary solutions and conformational landscapes. If successful, this study will contribute to the understanding of protein evolution and may find application in enzyme engineering for human purposes, by emphasizing the need to consider protein dynamics in directed evolution and enzyme function.

Rapid evolution of β-lactamase is an important cause of antibiotic resistance. Adaptation of the enzyme enables breakdown of new classes of β-lactam compounds and evasion of the effects β-lactamase inhibitors. Such inhibitors, in combination with β-lactam antibiotics, are used in the treatment of Gram negative pathogens and this treatment has also been proposed for infection by multi-resistant Mycobacterium tuberculosis strains. The aim of the project is to establish the evolutionary adaptability of the β-lactamase of M. tuberculosis, BlaC, in relation to inhibitors. The hypothesis is that the evasion of one inhibitor by mutagenesis of BlaC will make it much harder to also become resistant against a second inhibitor, because the enzyme also must remain sufficiently stable and catalytic active against β-lactam substrates. The cumulative cost of four selection pressures is expected to be too high to yield a viable enzyme. If this hypothesis is confirmed, it may be possible to identify pairs of inhibitors against which it is very difficult to develop resistance. The work will consist of three phases. (I) Generation of the variants and basic phenotypic characterisation using an Escherichia coli selection assay. (II) Characterisation of the structure, dynamics, stability and kinetic activity of the most interesting variants to elucidate the molecular basis for the observed phenotypes. (III) Testing of the properties of inhibitor resistant variants in a tuberculosis model system, granuloma formation in zebrafish embryos. The research will make it clear how easily resistance can evolve against the well-known inhibitors (clavulanic acid, tazobactam, sulbactam, avibactam) and whether resistance against more than one inhibitor can readily evolve. The underlying structural mechanisms will be established to provide a rationale for the observed effects. The biological assays will show whether in vitro enzyme phenotypes are expressed under close to physiological conditions.

This consortium aims to tackle a pressing issue in the Dutch society about how waste is used poorly, which leads to losing valuable materials, missing out on economic gains, and causing greenhouse gas emissions. To address this challenge and build a sustainable and thriving Dutch bioeconomy, we will develop innovative technology to turn low-quality waste into useful and sustainable materials that fit into a circular system. Our goal is to make the environment in the Netherlands cleaner and self-sustainable by cutting down on carbon emissions from fossil fuels, reducing critical materials in industry, reducing waste incineration, and re-using waste.

This application concerns a scientific meeting for which researchers working in the field of Proteostasis are invited. This concerns both researchers working in the chaperone field and researchers working in the ubiquitin field. Proteastasis is achieved through correct folding of proteins by chaperones and the degradation of misfolded proteins by the ubiquitin-proteasome system.