Abstract Reversible protein S-palmitoylation is an abundant posttranslational modification in the nervous system and is a key regulatory mechanism in numerous neuronal signaling pathways. Substrates for protein S- palmitoylation include neurotransmitter receptors and transporters, signal transducing GTPases, and synaptic scaffolds. Members of the DHHC family of protein acyltransferases mediate the addition of palmitate or other long-chain fatty acids to proteins and have been linked to a number of neurological disorders including intellectual disability and Huntington's Disease. The importance of S-palmitoylation as a regulatory modification in neuronal function and the alterations in S-palmitoylation associated with human disease warrant a mechanistic understanding of the enzymes that mediate this process. Accordingly, our long-term goals are to elucidate the molecular mechanisms underlying DHHC enzyme activity and its regulation by determining the first atomic resolution structure of a DHHC protein by x-ray crystallography. The goals of this proposal are to understand how self-association of DHHC proteins regulates their enzyme activity and to crystallize a DHHC protein. Using a bioluminescence resonance energy transfer assay in cells and in vitro enzyme assays with purified proteins, we determined that DHHC2 and DHHC3 proteins self-associate and that the extent of self-association tightly correlated with its enzyme activity. Furthermore, our pre-crystallization screening revealed that monodisperse and stable DHHC protein constructs exist predominantly as dimers. Based on these findings, we propose that DHHC PATs exist in a monomer-dimer equilibrium in cell membranes, where the monomeric form is active and the associated form is inactive. Testing this hypothesis is key to understanding how enzyme activity is regulated and critical for identifying conditions that will stabilize the protein for crystallization. We propose the following lines of investigation. (1) To address the activity states of the monomeric and dimeric forms of DHHC proteins, we will independently reconstitute DHHC monomers and dimers into nanodiscs and measure their enzyme activity. We will use single molecule photobleaching and total internal refection microscopy to determine the subunit stoichiometry of DHHC proteins expressed at the cell surface. (2) To identify conditions for the crystallization of a DHHC protein, we will optimize the DHHC construct using protein engineering. Folding- specific monoclonal antibodies will be generated and used to stabilize the construct. Crystallization conditions will be expanded to include the use of bicelles and lipidic cubic phases. Successful outcomes of these aims will greatly increase the chance of determining the much-needed first crystal structure of a DHHC protein, which we expect will become a strong foundation for understanding the molecular mechanisms of protein palmitoylation.