Molecular Switch between Structural Compaction and Thermodynamic Stability by the Xxx-Pro Interface in Transmembrane ß-Barrels.

Transmembrane ß-barrel scaffolds found in outer membrane proteins are formed and stabilized by a defined pattern of interstrand intraprotein H-bonds, in hydrophobic lipid bilayers. Introducing the conformationally constrained proline in ß-barrels can cause significant destabilization of these structural regions that require H-bonding, with proline additionally acting as a secondary structure breaker. Membrane protein ß-barrels are therefore expected to show poor tolerance to the presence of a transmembrane proline. Here, we assign a thermodynamic measure for the extent to which a single proline can be tolerated at the C-terminal interface of the model transmembrane ß-barrel PagP. We find that proline drastically destabilizes PagP by 7.0 kcal mol-1 with respect to wild-type PagP (F161 → P161). Interestingly, strategic modulation of the preceding residue can modify the measured energetics. Placing a hydrophobic or bulky side chain as the preceding residue increases the thermodynamic stability by ≤8.0 kcal mol-1. While polar substituents at the preceding residue decrease the PagP stability, these residues demonstrate stronger tertiary packing interactions in the barrel and retain the catalytic activity of native PagP. This biophysical interplay between enhanced thermodynamic stability and attaining a structurally compact functional ß-barrel scaffold highlights the detrimental effect caused by proline incorporation. Our findings also reveal alternative mechanisms that protein sequences can employ to salvage the structural integrity of transmembrane protein structures.