ИСТИНА

Due to hydrophobic nature membrane proteins remains “terra incognita” of structural biology for long time. This prevents rational investigation of the proteins at molecular level and effective structure-based drug design. Fibroblast growth factor receptor 3 (FGFR3) plays an important role in human development and diseases. There are mutations in the transmembrane region of FGFR3 (tmFGFR3), including point pathogenic mutations G380R and A391E, associating with human pathology states. We propose effective cell-free and bacterial expression systems that allow us to produce the target peptides in sufficient amounts for NMR structural studies. Interesting that for wild-type tmFGFR3 and for tmFGFR3(A391E) best yields were obtained using bacterial expression system whilst tmFGFR3(G380R) was best expressed in cell-free system. Purification procedures were developed for all peptides and isotope-labeled derivatives. Purified proteins were reconstituted into membrane-mimicking environment and characterized using dynamic light scattering, CD and NMR spectroscopy. High-resolution NMR structure of tmFGFR3 dimer was obtained. Some pathogenic mutations fall within the helix-helix interface and the others are within a putative alternative interface. Based on the obtained structure and other known data we propose a mechanism of the FGFR3-mediated signal transduction across the cellular membrane.