The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded β-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the β-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of β-sheet secondary structure and β-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of β-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.
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