Effect of the protein import machinery at the mitochondrial surface on precursor stability

Many biological processes require proteins to undergo conformational change s at the surface of membranes. For example, some precursor proteins unfold at the surface of mitochondria and chloroplasts before translocation into t he organelles, and toxins such as colicin A unfold to the molten globule st ate at bacterial surfaces before inserting into the cell membrane. It is co mmonly thought that the membrane surfaces and the associated protein machin ery destabilize the substrate proteins and that this effect is required for membrane insertion or translocation. One of the best characterized translo cation processes is protein import into mitochondria. By measuring the cont ributions of individual interactions within a model protein to its stabilit y at the mitochondrial surface and in free solution, we show here that the mitochondrial surface neither induces the molten globule state in this prot ein nor preferentially destabilizes any type of interaction (e.g., hydrogen bonds, nonpolar, etc.) within the protein. Because it is not possible to m easure absolute protein stability at the surface of mitochondria, we determ ined the stability of a tightly associated protein-protein complex at the m itochondrial import site as a model of the stability of a protein. We found the binding constants of the protein-protein complex at the mitochondrial surface and in free solution to be identical. Our results demonstrate that the mitochondrial surface does not destabilize importing precursor proteins in its vicinity.