One of the main goals of modern biology is the comprehension of protein operation mechanisms not only on the molecular level but also at the scale of spatial structure. Such data is essential to design rationally the proteins with desired properties, as well as to develop the targeted drugs. Particular attention is now being drawn to the membrane proteins, especially to the cell receptors, which play a major role in cell functioning. Despite the obvious importance, membrane proteins remain the insufficiently explored types of proteins. The main problem this project is assigned to solve is the investigation of Toll-like receptors signaling mechanism at the scale of protein structure. TLRs are the key players in the innate immunity system. Activation of TLRs leads to the immune response and inflammation, the family of receptors is involved in a number of diseases, including sepsis, immunodeficiency, atherosclerosis, and asthma. This makes TLRs the promising targets for the development of targeted medicines; more than twenty new drugs that affect TLRs are already in various phases of clinical trials. Despite the seeming clarity, many unanswered questions remain in the field of structural biology of toll-like receptors. In particular, the structures of the TIR domains of six members of the family are not obtained, the interfaces of the homo- and heterodimerization of the TIR domains for all receptors of the family, except for TLR10, are not described. The structures of the complexes between the TIR domains of TLRs and adapter proteins have not been studied, the structural organization of transmembrane domains and juxtamembrane regions is unclear for the majority of human TLRs. The effects that TLR domains impose on each other's structures are also not investigated. The project proposes to fill most of the listed gaps in the structural biology of TLRs: to investigate the structure of the TLR TIR domains in solution, determine the spatial structures of the transmembrane domains of several TLRs, investigate the interactions of TIR domains with Zn ions. We plan to localize the TLR TIR domain dimerization interfaces as well as the interfaces between the TIR domains of TLR and adapter proteins MyD88 and TIRAP. To investigate the effect of TLR dimerization on the interactions of their intracellular domains with the adapter proteins. The Project team utilizes high-resolution NMR spectroscopy to study the spatial structures of proteins. This method has its limitations, advantages, and disadvantages. Thus, NMR is able to study the behavior of the objects in solution, with the physiological parameters of the environment, while not only the structure but also the mobility of individual regions of the protein molecule can be investigated, which is an important piece of information to understand the functioning of cell receptors. On the other hand, there is a need to label the objects under investigation with the stable isotopes of nitrogen and carbon and membrane proteins need to be placed in a specialized membrane mimetic environment. Therefore, the bottleneck of most studies of eukaryotic proteins is the production of isotope-labeled proteins in bacterial cultures in the natively folded state. A substantial part of the Project will be devoted to the solution of this problem: we plan to develop approaches to the synthesis of intracellular and transmembrane domains of TLRs in the natively folded state in bacterial cultures, to develop the novel Lipodisc-type membrane mimetics to provide the adequate environment for the membrane proteins. Finally, throughout the Project, approaches will be developed to obtain the protein constructs containing the intracellular and transmembrane TLR domains. The most advanced technologies of protein ligation and segmental isotope labeling will be utilized for this purpose. Thus, the Project team aims to obtain important results in the field of protein engineering (development of technologies for the production of membrane proteins) and in the field of structural biology (obtaining the missing information on the structure of proteins, participants of TLR signaling cascades). All the results will be original and will not have analogs, the works will be performed on the most modern equipment using the most recent methods of multidimensional heteronuclear NMR spectroscopy in solution and will be published in peer-reviewed international journals.