title: Effect of the protein import machinery at the mitochondrial surface on precursor stability.
abstract: Many biological processes require proteins to undergo conformational changes at the surface of membranes. For example, some precursor proteins unfold at the surface of mitochondria and chloroplasts before translocation into the organelles, and toxins such as colicin A unfold to the molten globule state at bacterial surfaces before inserting into the cell membrane. It is commonly thought that the membrane surfaces and the associated protein machinery destabilize the substrate proteins and that this effect is required for membrane insertion or translocation. One of the best characterized translocation processes is protein import into mitochondria. By measuring the contributions of individual interactions within a model protein to its stability at the mitochondrial surface and in free solution, we show here that the mitochondrial surface neither induces the molten globule state in this protein nor preferentially destabilizes any type of interaction (e.g., hydrogen bonds, nonpolar, etc.) within the protein. Because it is not possible to measure absolute protein stability at the surface of mitochondria, we determined the stability of a tightly associated protein-protein complex at the mitochondrial 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.
authors.affiliation: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208-3500, USA.
authors.forename: S
authors.initials: S
authors.lastname: Huang
authors.forename: S
authors.initials: S
authors.lastname: Murphy
authors.forename: A
authors.initials: A
authors.lastname: Matouschek
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D004795:Enzyme Stability; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D011498:Protein Precursors; D011994:Recombinant Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease