Miłosz Ruszkowski Structural and biochemical studies on proteins involved in hormonal regulation in plants. The thesis describes studies on a few proteins that are involved in hormone signal transduction in plants or are able to bind phytohormones. Medicago truncatula was chosen as the source organism for all of the examined proteins as it is nowadays considered a model legume plant. Legume plants are characterized by a particular symbiotic interaction with nitrogen-fixing bacteria in special organs on roots (nodules). Legumes and nodulation are important in context of one of the presented results. The target proteins were mostly studied with the use of macromolecular crystallography, and the obtained results were verified by other methods. Considerable part of the thesis concerns the proteins that are involved in hormone signal transduction. The first protein of interest was histidine-containing phosphotransfer protein (MtHPt1). The crystal structure was solved with high resolution data (1.45 Å). The overall fold of MtHPt1 is similar to HPt models from other plants. It has also been compared to the proteins from this particular family, originating from different kingdoms. The model of MtHPt1 has been, so far, the only crystal structure of a plant HPt protein proven to be active in a biochemical assay in vitro. At this point, it is necessary to mention the intracellular moiety of the cytokinin receptor (MtCRE1’) that was produced within the scope of the thesis and used to successfully verify the biological significance of MtHPt1. The fact that MtHPt1 can be activated by MtCRE1’ in the presence of ATP confirms the MtHPt1 as a part of the cytokinin signal transduction pathway. The 3D structure of MtCRE1’ itself or in complex with MtHPt1 remains unsolved despite significant effort. Another protein from HPt family (MtHPt2) was also studied in this thesis. The crystal structure of MtHPt2 was determined with subatomic resolution data (0.92 Å). Data of this quality are very rarely obtained in macromolecular crystallography, and the presented model was refined against one of the highest resolution data as far as plant proteins are concerned. It is, however, still a pending question in which phosphorylation cascade MtHPt2 takes part in. The thesis is also focused on the Nodulin 13 (MtN13). The protein was found to be expressed specifically in the root nodules (Gamas et al., 1998). It is, therefore, a nodulation marker, but its exact function remains unrevealed. MtN13 belongs to pathogenesis-related proteins class 10 (PR-10). This work describes interaction of MtN13 with cytokinins, a class of plant hormones that are involved in nodulation of legumes. Two exciting results were obtained within the work of the dissertation. Firstly, MtN13 can bind different natural cytokinins in a conserved and apparently specific manner. A specificity like this has never been observed for a PR-10 protein, and the only example that binds cytokinins so tightly is the cytokinin receptor characterized structurally for A. thaliana. Secondly, cytokinin binding by MtN13 is directly related with dimerization of the protein. More precisely, a loop from one protein molecule takes part in binding of the cytokinin in the other MtN13 molecule. The loops are swapped between the protein entities and the dimer contains two protein molecules and two cytokinin ligands. These results are extremely interesting in context of nodulation of legumes as MtN13 can interfere with the nodulation pathways by regulating the concentration of available cytokinins. However, extensive in planta studies are required to verify this hypothesis.