Energetics of side-chain partitioning of β-signal residues in unassisted folding of a transmembrane β-barrel protein

Abstract

The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane β-barrels form the β-signal motif required for assisted β-barrel assembly in vivo but are believed to be less important for β-barrel assembly in vitro. Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the β-signal motif to the unassisted folding of the model β-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the β-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain–specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP β-barrel scaffold. We conclude that the PagP C-terminal β-signal motif influences the folding cooperativity and stability of the folded β-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein β-signal motif depend on the nature of the amino acid side chain.

Hydrophobic Characteristic Is Energetically Preferred for Cysteine in a Model Membrane Protein

“…PagP possesses 12 tryptophans that are distributed throughout the protein. We and others have observed that PagP exhibits hysteresis or incomplete unfolding in alkaline conditions in lipid vesicles ( 20 , 22 ). Hence, we examined only the folding titration of the equilibrium reaction in small unilamellar vesicles (SUVs) of 1,2-dilauroyl- sn -glycero-3-phosphocholine (DLPC), as reported previously ( 20 ).…”

“…We and others have observed that PagP exhibits hysteresis or incomplete unfolding in alkaline conditions in lipid vesicles ( 20 , 22 ). Hence, we examined only the folding titration of the equilibrium reaction in small unilamellar vesicles (SUVs) of 1,2-dilauroyl- sn -glycero-3-phosphocholine (DLPC), as reported previously ( 20 ). Briefly, we added 5 μ g/ μ L (0.26 mM) unfolded PagP in a 1:9 ratio to 8.9 mM DLPC SUVs to generate a folding stock containing 26 μ M protein and 8 mM lipid dissolved in 4.5 M GdnHCl prepared in 20 mM Tris-Cl (pH 9.5) and 20 mM 1,4-dithiothreitol (DTT).…”

“…The eight-stranded β -barrel, PagP, has been widely used as a model for membrane protein folding studies owing to its ability to spontaneously fold into an array of membrane mimetics ranging from detergent micelles to lipidic vesicles ( 20 , 22 , 26 ). Here, we generated a library of single-cysteine mutants (native PagP from E. coli is devoid of cysteine residues) by strategically choosing 27 lipid-facing host sites near the water-lipid interface and toward the midplane on each strand of the eight-stranded transmembrane barrel ( Fig.…”

“…E. coli BL21(DE3) cells were transformed with these plasmid constructs and used for protein production. Proteins were expressed in the form of inclusion bodies and processed to ∼95% purity using reported methods ( 20 ).…”

“…To this end, we employed a mutational strategy that involved introducing cysteines in place of a total of 27 lipid-facing residues located toward the midplane as well as near the interface region of each of the eight β -strands. Along similar lines, we used a lipid bilayer system consisting of phosphatidylcholine vesicles to create a near-native environment to accommodate PagP, as opposed to our previous studies with membrane mimetics such as detergent micelles ( 20 , 21 ). This membrane system now recreates the chemical environment of the biological membrane by providing a polarity gradient across the bilayer and, at the same time, possesses the physical properties of membrane lipids such as lateral bilayer pressure.…”