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Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers.

Journal of molecular biology (2011-09-06)
C Preston Moon, Sarah Kwon, Karen G Fleming
RÉSUMÉ

The free energy of unfolding of a membrane protein from lipids into water (ΔG(o)(w,l)) describes its equilibrium thermodynamic stability. Knowing this parameter gives insight into a membrane protein's sequence-structure-energy relationships. However, there are few measures of membrane protein stability because of the technical difficulties associated with unfolded and partially folded states. Here, we describe the experimental process that allowed us to measure the ΔG(o)(w,l) of the outer membrane phospholipase A into large unilamellar vesicles (LUVs) of 1,2-dilauroyl-sn-glycero-3-phosphocholine. To arrive at this reversible folding condition, we screened a large number of experimental variables: temperature, incubation time, salt concentration, pH, lipid composition and liposome morphology. The principal challenge we encountered under most conditions was hysteresis between folding and unfolding titrations. A second factor that compromised reversible folding was the observation that a fraction of the protein population tended to aggregate. We found that hysteresis could be completely eliminated on a feasible timescale by conducting experiments at acidic pH, by the slow dilution of the protein in the initial titration setup and by utilizing a low concentration of a detergent as a temporary "holdase" to solubilize the protein upon its initial dilution into folding conditions. We confirmed that the detergent did not disrupt the LUVs using fluorescence emission of lipid-sensitive dyes and light scattering. The results of our parameter search should be generally useful for efforts to measure ΔG(o)(w,l) for other membrane proteins.

MATÉRIAUX
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Description du produit

Sigma-Aldrich
Phospholipase A1 from Aspergillus oryzae
Sigma-Aldrich
1,2-Didodecanoyl-sn-glycero-3-phosphocholine, ≥99%, synthetic
Avanti
12:0 PS, 1,2-dilauroyl-sn-glycero-3-phospho-L-serine (sodium salt), powder