Mitochondria, also known as the powerhouses of the cell, are organelles that provide cells with chemical energy essential for anabolic biochemical processes. In response to changing energy needs of the cell, mitochondria being dynamic in nature undergo fission and fusion. Mitochondrial fission is dependent on GTPase dynamin-related protein 1 (DRP1, isoforms 1-9). DRP1 also plays a crucial role in cell differentiation, immunity, apoptosis, signaling, metabolism, and aging. The crystal structure of DRP1 Isoform 2 has been solved using x-ray crystallography. However, the ~100 amino acid long variable domain containing regulatory post-translational modifications (PTMs) remains unresolved. Mitochondrial fusion is coordinated by mitofusin 1 and 2 (Mfn1 and Mfn2) for the outer membrane and Optic atrophy protein 1 (OPA1) regulates the fusion of the inner mitochondrial membrane. OPA1 mutant cells derived from patients exhibit defects in mitochondrial architecture, mtDNA copy number, cristae structure, oxidative phosphorylation, Calcium buffering, ATP synthesis, mitophagy, and cell survival. Our preliminary data indicate that long-chain fatty acid-CoA esters (FACE) allosterically activate DRP1 isoform 3, regulating mitochondrial fission and morphology. In this study, we will use palmitoyl-CoA to stabilize the variable domain of DRP1 and elucidate its structure. Furthermore, we will produce site-specific acetylated OPA1 proteins using genetic code expansion, perform a GTP hydrolysis assay to determine the OPA1 enzymatic activity, test the functional consequence of site-specific acetylation on OPA1 lipid binding and oligomerization, and elucidate the structure of human OPA1 in the presence and absence of GTP analogs using cryo-EM and single particle analysis.