Thermodynamic studies of the insertion and folding of membrane proteins: yeast cytochrome c oxidase

Yeast cytochrome c oxidase is a multisubunit protein located in the inner mitochondrial membrane where it serves as the final intermediary in the respiratory chain and as a proton pump. The three largest subunits are synthesized within the mitochondria and the remaining six subunits are encoded by the nuclear genome and then imported to the inner mitochondrial membrane from the cytoplasm. The exact mechanism of assembly and stabilization of this protein complex is only beginning to be elucidated. In order to investigate the stabilizing interactions within the enzyme complex and the mode of association between subunits we have performed structural stability and thermodynamic perturbation experiments using different biophysical techniques including high sensitivity differential scanning calorimetry and differential solubility thermal gel analysis. These studies have suggested the existence of hierarchical layers of interactions between subunits that define three important cooperative structures. The major components of these structures in the yeast enzyme appear to be: a) subunits I and II; b) subunit III; and, c) the imported subunits IV and VI. In order to confirm these assignments and study directly the assembly of the protein complex, subunit dissociation experiments under controlled conditions have been performed.