Elucidating the Folding Pathway of Outer Membrane Protein A

Understanding how a protein folds to its functional structure is a central question of biophysics that is still not completely understood. Proteins that reside in membranes face the additional complication of inserting into the lipid bilayer while forming the correct conformation. Membrane proteins are essential for a number of biological processes and many diseases are linked to misfolding of these proteins, so gaining a better understanding of how membrane proteins attain their native structures is of great importance to biomedical research. Although most membrane proteins in the cell require the assistance of folding machinery to be properly folded and inserted, β-barrel outer membrane proteins are capable of spontaneously folding to the native state in the absence of folding factors, indicating an intrinsic folding mechanism dependent only on the amino acid sequence and the membrane environment. We have investigated several aspects of the intrinsic folding pathway for the model bacterial outer membrane protein OmpA, including the conformation and interactions of the aqueous unfolded state, the extent of secondary structure formation during the folding and insertion process, and the presence of off-pathway intermediates. In studying the conformations of the unfolded state, we found that the periplasmic domain of the protein, which has typically been ignored in folding studies, behaves as an independent folding unit and helps reduce the self-association of the unfolded transmembrane barrel domain. We also determined that the unfolded barrel domain has no regular structure and an expanded conformation, indicating that structure can only form upon interaction with a membrane. We next investigated the folding and membrane insertion of the OmpA β-barrel and developed a comprehensive kinetic model to describe the pathways and folding intermediates of the protein. CD measurements revealed a partially inserted, penultimate state with a higher content of β-sheet structure than the native state. Based on our data and previous work we have proposed a detailed folding model for β-barrel proteins that is facilitated by the presence of defects in the lipid bilayer and secondary structure formation in the protein. We also identified several off-pathway intermediate states that give rise to additional exponential phases in the data