Malleability of the folding mechanism of the outer membrane protein PagP: parallel pathways and the effect of membrane elasticity.

Understanding the interactions between membrane proteins and the lipid bilayer is key to increasing our ability to predict and tailor the folding mechanism, structure and stability of membrane proteins. Here, we have investigated the effects of changing the membrane composition and the relative concentrations of protein and lipid on the folding mechanism of the bacterial outer membrane protein PagP. The folding pathway, monitored by tryptophan fluorescence, was found to be characterized by a burst phase, representing PagP adsorption to the liposome surface, followed by a time course that reflects the folding and insertion of the protein into the membrane. In 1,2-dilauroyl-sn-glycero-3-phosphocholine (diC(12:0)PC) liposomes, the post-adsorption time course fits well to a single exponential at high lipid-to-protein ratios (LPRs), but at low LPRs, a second exponential phase with a slower folding rate constant is observed. Interrupted refolding assays demonstrated that the two exponential phases reflect the presence of parallel folding pathways. Partitioning between these pathways was found to be modulated by the elastic properties of the membrane. Folding into mixed 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine:diC(12:0)PC liposomes resulted in a decrease in PagP adsorption to the liposomes and a switch to the slower folding pathway. By contrast, inclusion of 1,2-dilauroyl-sn-glycero-3-phosphoserine into diC(12:0)PC liposomes resulted in a decrease in the folding rate of the fast pathway. The results highlight the effect of lipid composition in tailoring the folding mechanism of a membrane protein, revealing that membrane proteins have access to multiple, competing folding routes to a unique native structure.

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