Rab3A is the most abundant Rab family protein found in the mammalian nervous system. It is a small GTPase, membrane-anchored protein involved in the regulation of vesicular transport and Ca2+-induced exocytosis of neurotransmitters and hormones. Once Rab3A is expressed in vivo, its C-terminal linker tail is lipidated by a covalent attachment of two hydrophobic isoprenyl moieties. Lipidated Rab3A is then delivered to the surface of a synaptic vesicle where lipid moieties embed into a phospholipid bilayer and anchor the protein to the membrane. The head domain contains the catalytic and functional segments of the protein, by which Rab3A is able to bind GTP nucleotide, change structural conformation and interact with a number of important effector proteins involved in vesicular transport and exocytosis. One such Rab3A effector is alpha-synuclein (αS) which is a small intrinsically disordered protein associated with progression of Parkinson's disease. αS was found to interact with a membrane-anchored GTP-bound Rab3A in vivo and implicated in regulation of Rab3A functionality leading to disruption of synaptic vesicle exocytosis.
The work presented here seeks to re-create a near native Rab3A and αS environment in vitro in hope to detect their interaction and elucidate its structural mechanism. The study describes the development of a synthetic Rab3A-SUV model system which could be used for functional and structural protein interaction studies of Rab3A and its effectors. A combination of solution and solid-state nuclear magnetic resonance as well as other functional and structural techniques such as dynamic light scattering, circular dichroism, thioflavin T assay and cryo-EM were used to assess the importance of phospholipid membrane in Rab3A functionality. It was demonstrated that the presence of a membrane significantly affects Rab3A functionality. Therefore, future studies working on Rab3A and its protein effectors must take this into account and ideally use at least some type of phospholipid bilayer mimetic.