DNA-MolScaff · Reconstitution of Crista Junction proteins using DNA origami-based Molecular Scaffolds for Structural and Functional analyses

Mitochondrial cristae are functional dynamic compartments containing the respiratory chain complexes where oxidative phosphorylation occurs. They are separated from the inner boundary membrane by highly curved and tightly regulated membrane compartments called crista junctions (CJs). A key multi-protein complex, mitochondrial contact site and cristae organizing system (MICOS), is enriched in CJs and plays a central role in modulating the architecture and function of CJs. Recently, a MICOS subcomplex built by the Mic60-Mic19 tetramer was proposed to traverse the CJ like a molecular strut and hypothesized to act as a diffusion barrier for metabolites and proteins between the inner mitochondrial membrane and the cristae lumen. However, the exact location of this MICOS subcomplex in CJs, its proposed diffusion barrier function and its detailed role in modulating CJ architecture are not clear. Here, I propose to fabricate a CJ mimic to reconstitute the Mic60-Mic19 tetramer into an octagonal donut-shaped DNA origami scaffold. The surface of the DNA origami will be coated with a lipid monolayer, enabling stabilization of the reconstituted complex, and facilitating structural studies using cryo-electron microscopy (cryo-EM). Subsequently, the role of Mic60-Mic19 complex and its intrinsically disordered regions as a diffusion barrier will be probed by incorporating the CJ mimics onto the surface of the liposomes. These efforts will reveal the structure of the Mic60-Mic19 subcomplex in a near-native environment and its role in the transport of proteins and metabolites, thereby providing an in-depth understanding of its cellular function. The realization of membrane-coated DNA origami molecular scaffolds as platforms for reconstituting proteins may pave the way for nano-tools that are made from DNA for structural and functional exploration of other challenging membrane and membrane-interacting proteins, which could yield valuable insights into key biological processes.