The accuracy of protein synthesis. The research aims in this area are to understand the molecular basis and the biological importance of cellular mechanisms that ensure the accuracy of protein biosynthesis (translation). Erroneously synthesized proteins affect cell viability and function and are therefore associated with cellular aging and the development of neurodegenerative diseases. Using biophysical, proteomic, biochemical, computational and genetic analysis of structure/function relationship of aminoacyl-tRNA synthetases (AARSs), the key proteins that control the accuracy of protein biosynthesis, together with analysis of kinetic models of AARSs catalytic functions, we shed more light on the mechanistic details, evolution and biological relevance of proofreading reactions of these enzymes, which are developed to ensure the appropriate level of translational accuracy. To understand the impact of the inaccurate protein biosynthesis on protein structure/function we will produce the mistranslated model proteins. Understanding of these processes provides a platform for the synthesis of inhibitors with pharmacological activity, additional components for multicomponent antibiotics, and the treatment of diseases associated with mistranslated proteins. Our research is highly competitive and it is conducted in collaboration with prominent international groups that have complementary expertise (structural biology, laboratory-directed protein evolution, bioinformatics). Research in the field of the accuracy in protein biosynthesis will also include proteomic research in order to analyze and understand the relevance or permissibility of translational accuracy on a global scale. The long-term goal is to include proteomic studies based on mass spectrometry (LC-MS / MS) and to obtain the required instrumentation at the Department of Biochemistry since modern biochemical research is increasingly dependent on this aspect.
Design of Proteins with New Properties. Research goals are to create proteins with novel/improved properties that have superior biotechnological or biomedical applications using methods of protein engineering. Moreover, protein redesign has important application in biochemical, biophysical and cellular-biological studies as it enables the creation of specific properties that can be used to analyze the structure and function of the protein and to track their localization in the cell. Research is focused on the incorporation of synthetic (artificial) amino acids into proteins to effectively extend the standard amino acid alphabet. Interesting candidates to expand Nature's alphabet are fluorinated amino acids because fluorinated therapeutic peptides can easily pass through the membrane and thus have a better pharmacological effect. On the other hand, an attractive biotechnological line of research may include redesigning the existing protein biosynthesis system to prevent incorporation of non-proteinogenic amino acids that accumulate in the cell. This problem is recognized in the process of production of therapeutic proteins in bacterial cells and as such poses a serious problem in the pharmaceutical industry. The research is international, multidisciplinary and is at the intersection of biochemistry, synthetic biology and biotechnology.
Aminoacyl-tRNA-synthetases as targets for antibiotics. The research goals in the field of antibiotic mechanisms of action are to shed light on the correlation between the structure/function of aminoacyl-tRNA synthetases (AARS) and their role of a target in the production of natural antibiotics and in the development of synthetic drugs. Emphasis is on the research related to antibiotic resistance, which is one of the biggest global health problem. Mupirocin is an antibiotic that inhibits protein biosynthesis by inhibition of isoleucyl-tRNA-synthetase (IleRS). Mupirocin resistance is a common problem among the hospital strains of the pathogenic Staphylococcus aureus. Resistance in hospital strains, but also in nature, develops through the evolution of IleRS that is less sensitive to mupirocin or through the acceptance of an additional IleRS, which is insensitive to mupirocin. Using biochemical, kinetic, computational and genetic research in collaboration with groups dealing with biochemical evolution and structural biology, the goal is to address the key issues related to the mechanism of action and resistance to antibiotics as well as the connection of this process with the accuracy of the translation.
Novel noncanonical functions of aminoacyl-tRNA synthetase. Aminoacyl-tRNA synthetases often have additional functions in the cell and participate in a variety of cellular processes that are not directly related to protein biosynthesis. Noncanonical function studies will focus on poorly explored plant aminoacyl-tRNA synthetases, and will examine their role in cell response to abiotic stress using biochemical, molecular biology and genetic methods. At the same time, the protein interactors and the biological role of macromolecular complexes involving plant aminoacyl-tRNA synthetases and their influence on the growth and development of plants will be investigated. The subject of the study will be also amino acid:[carrier protein] ligases, bacterial homologues of aminoacyl-tRNA synthetases that evolved a new and unexpected substrate specificity towards aminoacylation of the carrier proteins instead of tRNAs. Their biological role is unknown, they do not participate in protein biosynthesis, but most likely in the biosynthesis of secondary metabolites or antibiotics.
Interactions of proteins with other biomolecules. Specific interactions between molecules are fundamental to all biological processes. To understand complex interacting systems in the cell, protein-protein interactions, interactions between protein and nucleic acids or with small ligands will be monitored. Various techniques and methods for analyzing macromolecular interactions will be used, with emphasis on quantitative determination of the binding free energy (thermophoresis, fluorescence spectrometry, isothermal titration calorimetry). Research has potential applications in biotechnology and biomedicine since many drugs act as inhibitors and modulators of biomacromolecules and their complexes.
Computational methods in biochemical research. Modern (bio)chemical research implies the use of computational methods in everyday work. Computational methods are used to study the structure, dynamics and energetics at the atomic level of biological macromolecules such as proteins, nucleic acids, and their complexes as well as their complexes with small compounds. In the absence of experimentally solved structures, computational methods are also used for predicting protein structures based on their amino acid sequence. Prediction of protein structures has been advanced in recent years because of methodological development and the exponentially growing number of experimentally solved structures that are used as structural templates. Therefore, computational methods complement experimental research and can significantly contribute to the following objectives: structure and function of erroneously synthesized model proteins and proteins with incorporated synthetic amino acids, modeling of aminoacyl-tRNA synthetases (AARS) to understand the mupirocin resistance. In addition to AARS-related research, computational methods will be used for modeling structure, function, dynamics, studying protein interaction networks and the catalytic mechanism of different enzymes involved in various metabolic pathways. Computational methods of different complexity such as molecular mechanics, molecular dynamics, quantum mechanical methods as well as their mutual combination and empirical valence bond (EVB) simulations will be used in the research.