Studies on the lipid dependency and mechanism of the translocation of the mitochondrial precursor protein apocytochrome c across model membranes

The lipid dependency of apocytochrome c binding to model membranes and of the translocation of the precursor protein across these membranes was studied by using large unilamellar, trypsin-containing vesicles. These vesicles were improved with respect to those used in a previous article (Rietveld, A., and de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6706), in the sense that a lower amount of trypsin was enclosed. In mixed egg phosphatidylcholine/bovine brain phosphatidylserine vesicles, both the Kd of apocytochrome c binding (about 20 microM) and the number of phosphatidylserine molecules interacting with the protein was found to be constant. When the phosphatidylserine fraction in the vesicles is more than 15-30% apocytochrome c addition results in the exposure of (a part of) the protein to the internal, trypsin-containing vesicle medium, which process we conceive as a translocation event. Also the interaction of apocytochrome c with vesicles composed of phosphatidylcholine and another acidic phospholipid in a 1:1 ratio, leads to the translocation of the protein across the model membrane. The affinity of this binding was found to be in the order cardiolipin greater than phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. By varying the lipid composition of the vesicles, it could be demonstrated that the translocation requires a fluid bilayer. In addition, protein specificity was shown for the translocation process. Although apocytochrome c-lipid interaction causes vesicle aggregation, fusion by lipid mixing could not be detected. Due to the apocytochrome c-lipid interaction also, protein aggregates and oligomers have been formed. These results will be discussed in the light of a model for translocation of a precursor protein across a model membrane. The relevance for the mitochondrial system will also be discussed.     

Recursive use of evolutionary conservation data in molecular modeling of membrane proteins A model of the multidrug H+ antiporter emrE.

Membrane proteins are currently the most biomedically important family of proteins, serving as targets for the majority of pharmaceutical agents. It is also clear that they are invariably abundant in all of the genomes sequence so far, representing up to a third of all open reading frames. Finally, and regrettably, it is clear that they are highly resistant to structural elucidation, representing less than 0.2% of the Protein Data Bank. Recent accomplishments in genome sequencing efforts, however, may help offset this imbalance through the availability of evolutionary conservation data. Herein, we develop a novel approach, utilizing a combination of evolutionary conservation data and global searching molecular dynamics simulations to model membrane proteins, deriving a model for the multidrug H+ antiporter EmrE, a transmembrane four-helix bundle. Structures resulting from an extensive, rotational molecular dynamics search, were evaluated by comparing the residue specific interaction energy and the evolutionary conservation data. Subsequent rounds of molecular dynamics, in which confinement of the search space was undertaken in order to achieve a self consistent result, point to a structure that best satisfies the evolutionary conservation data. As the conservation patterns calculated for each of the helices suggested that the different conservation pattern for helix 3 (as well as being the most conserved) might be due to the oligomeric nature of EmrE, a dodecamer of helices was constructed based on the result of a search of helix 3 as a trimer. The resulting interaction energy per residue in the final model is in reasonable agreement with the evolutionary data and consistent with recent site directed mutagenesis experiments, pointing to the strength of this method as a general tool.