Dynamin: Ligand Interactions and Conformational Dynamics

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Dynamin is a large molecular weight (~98kDa; 870 amino acids) GTPase, which is an important component of a wide variety of biological processes which are critical for cell viability. In mammals, dynamin is encoded by three conventional dynamin genes (Dyn1, Dyn2, Dyn3). Dyn1 is expressed in neurons, Dyn2 is ubiquitously expressed, and Dyn3 is most highly expressed in testes but is also detectable in neurons and lung. Purified recombinant dynamin can self-assemble into rings and spirals, which suggests that all the necessary binding interactions are contained within the protein sequence. Both dynamins 1and 2 exist in equilibria involving monomeric and multimeric species and we hypothesis that this lower-order oligomerization may represent a novel site of regulation. In this proposal, we plan to use fluorescence polarization and fluorescence correlation spectroscopy (PCS) to study the self-association of dynamin in vitro and to determine the effects of bound nucleotide and lipid on this equilibrium. We shall also extend these studies to in vivo conditions by utilizing an EGFP construct of dynamin. We shall use PCSto detect association of dynamin and endophilin in vivo and to study the effects of various mutations (in both dynamin and endophilin) on this association. Earlier, we used a giant unilammelar vesicle system (GUV) to directly observe the interaction of fluorescently-labeled dynamin with different lipid systems. These studies suggested the value of the GUV system for direct information on the interaction of dynamin with phospholids. To continue these studies, we proposeto use PCSto to study dynamin 2 labeled with small fluorophores as well as EGFP-constructs, associating with GUVs. We further hypothesize that GTP release is slowed when dynamin is bound to a scaffold, e.g., anionic liposomes or microtubules, prior to polymerization. To test this possibility we will use stopped-flow kinetic methods to measure nucleotide binding and release in the presence of PIP2/PC liposomes. These studies will provide quantitative information on the strengths and significance of dynamin's interactions.and on aspects of its conformaitonal dynamics. Since dynamin is fundamental to diverse and critical biological functions, including receptor mediated endocytosis (involved in cholesterol uptake) and synaptic vesicle recycling (important in nerve conduction), this information is significant to public health.