Weibel-Palade bodies (WPBs) are endothelial-specific secretory vesicles that serve a role of vascular ‘emergency kit’.1 WPBs store haemostatic and pro-inflammatory mediators that can be released on demand, enabling rapid response to vascular injury. However, untimely and inappropriate exocytosis of WPBs can contribute to the development and progression of atherosclerosis, and thus secretion from endothelial cells has to be a tightly coordinated process. Rab GTPases are important regulators of all aspects of intracellular membrane trafficking. Rab46 is a novel multi-domain Rab GTPase that localizes to WPBs.2 In addition to the conserved Rab domain, Rab46 contains a coiled-coil domain responsible for interactions with other proteins and EF-hand domain that acts as a built in Ca2+ sensor. Recently Rab46 has been shown to act directly as dynein adaptor in T cells.3 Moreover, findings from our lab demonstrated that Rab46 is a key regulator of context-dependent differential WPB cargo secretion. As the conformational changes observed during activation and inactivation of Rab proteins might provide binding pockets that could be modulated by small molecules, an understanding of the 3D atomic structure of Rab46 could provide novel therapeutic targets for cardiovascular disease. Since no structural studies on Rab46 have been performed to date, this project aims to investigate the structure, folding and dynamic properties of Rab46. 3D structures for the EF-hand, coiled-coil and Rab domains of Rab46 were predicted and analysed using protein structure prediction servers (I-TASSER and Phyre2) and Maestro software. Full-length protein and its individual functional domains were cloned into pET32a-LIC expression vector using In-Fusion® Cloning system. The histidine-tagged recombinant proteins were expressed in BL21 and Rosetta 2 E. coli strains and purified by immobilized metal ion affinity chromatography, followed by size exclusion chromatography allowing to obtain high purity protein at a final concentration of 0.3 mM, suitable for natural abundance 15N HSQC NMR experiments. Future work will focus on Rab46 structure elucidation using low resolution structural and biophysical techniques. Isotopically-labelled recombinant proteins will be produced for investigation of the dynamics and inter-domain interactions of Rab46 using high-resolution solution NMR. Structures of the domains obtained from homology modelling will be investigated to identify functionally important residues and guide new experiments such as site-directed mutagenesis. Moreover, coiled-coil domains of other dynein adaptors will be analysed using in silico approaches to explore the nature of the interaction between Rab46 and dynein. Ultimately, in-cell NMR will be used to observe conformational changes in response to agonists in the native environment.