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

Project description

Molecular insight into mitochondrial function

Mitochondria are known as the ‘powerhouses’ of the cell, primarily providing energy for various cellular processes. They possess a unique structure that includes an outer membrane, and an inner membrane folded into cristae where oxidative phosphorylation takes place. Mitochondrial cristae are separated from the inner boundary membrane by regions called cristae junctions (CJs) whose shape and function are regulated by a protein complex. With the support of the Marie Skłodowska-Curie Actions programme, the DNA-MolScaff project will investigate the movement of metabolites and proteins between the mitochondria inner membrane and the cristae. With the use of DNA origami, the research team will create a CJ model and study specific proteins, improving our understanding of mitochondrial function.

Objective

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.

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Coordinator

MAX DELBRUECK CENTRUM FUER MOLEKULARE MEDIZIN IN DER HELMHOLTZ-GEMEINSCHAFT (MDC)
Net EU contribution
€ 173 847,36
Address
ROBERT ROSSLE STRASSE 10
13125 Berlin
Germany

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Region
Berlin Berlin Berlin
Activity type
Research Organisations
Links
Total cost
No data