Electron Transport Chain Complex II Regulates Steroid Metabolism.

The first steroidogenic enzyme, cytochrome P450-side-chain-cleavage (SCC), requires electron transport chain (ETC) complexes III and IV to initiate steroid metabolic processes for mammalian survival. ETC complex II, containing succinate dehydrogenase (quinone), acts with the TCA cycle and has no proton pumping capacity. We show that complex II is required for SCC activation through the proton pump, generating an intermediate state for addition of phosphate by succinate. Phosphate anions in the presence of succinate form a stable mitochondrial complex with higher enthalpy (-ΔH) and enhanced activity. Inhibition of succinate action prevents SCC processing at the intermediate state and ablates activity and mitochondrial protein network. This is the first report directly showing that a protein intermediate state is activated by succinate, facilitating the ETC complex II to interact with complexes III and IV for continued mitochondrial metabolic process, suggesting complex II is essential for steroid metabolism regulation.

The role of membrane properties in Mistic folding and dimerisation.

Membranes not only provide cellular compartmentalization but influence protein behavior and folding by virtue of the multitude of different lipid types. We have studied the impact of lipid composition on the folding of the membrane-associated protein Mistic from B. subtilis. We use dimerisation via the single Cys3 residue as monitor for the degree of correct folding, since mis- or unfolding will expose the otherwise buried Cys3. We find great variability in how lipids affect protein production and dimerization, ranging from high production and low dimerization via increased production and higher dimerization to low production and low dimerization. Phosphocholine (PC) vesicles, in particular di-oleoyl-PC, lead to the highest production levels. Shorter chain lengths lead to reduced production but higher levels of dimerization. Different lipids may promote correct folding of Mistic to different extents, mediated by proper hydrophobic matching (attained for long-chain but not short-chain PC vesicles) and the existence of a fluid phase (the gel phase reduces production as well as dimerization, probably by immobilizing Mistic on the surface). The very fact that different lipids have an effect indicates that Mistic behaves like a bona fide membrane protein with a clear preference for membranes of a certain thickness and flexibility.

Cell-free synthesis and folding of transmembrane OmpA reveals higher order structures and premature truncations.

We use a cell-free transcription-translation system to monitor the effect of different lipids on the synthesis and folding of the transmembrane domain of the outer membrane protein OmpA from E. coli under physiological conditions. Folding is consistent with previous observations made in vitro at high pH. Synthesis and folding yields are optimal in phosphocholine lipids, particularly in short chain lipids and small vesicles, while lipid rafts do not promote folding compared to the folding in the absence of lipids. Truncated species are observed during translation in the presence of the periplasmic chaperone Skp, which likely binds to the newly synthesized polypeptide chain during cell-free translation and thus prematurely terminate polypeptide chain synthesis. In contrast, folded and unfolded dimers of OmpA correlate negatively with folding yields. This suggests that dimer formation competes with folding and insertion of monomeric OmpA, though folded dimers slowly appear to convert to folded monomers.