The ability of histidine to participate in a wide range of stabilizing polar interactions preferentially populates this residue in functionally important sites of proteins. Histidine possesses an amphiphilic and electrostatic nature that is essential for amino acids residing at membrane interfaces. However, the frequency of occurrence of histidine at membrane interfaces, particularly transmembrane β-barrels, is lower than those of other aromatic residues. Here, we carry out comprehensive energetic measurements using equilibrium folding of the outer membrane enzyme PagP to address the contribution of a C-terminal interface histidine to barrel stability. We show that placing histidine at the C-terminus universally destabilizes PagP by 4.0–8.0 kcal mol–1 irrespective of the neighboring residue. Spectroscopic and electrophoretic measurements indicate that the altered stability may arise from a loss of barrel compaction. Isoleucine, methionine, and valine salvage this destabilization marginally (in addition to tyrosine, which shows an exceptionally high folding free energy value), when placed at the penultimate position, at the expense of an altered folding pathway. Double-mutant cycle analysis indicates that the coupling energy between the terminal and penultimate residues in PagP-X160H161 increases when the level of intrinsic destabilization by the terminal H161 is high. Our observations that neighboring residues cannot salvage the energetic destabilization of histidine may explain why histidine is less abundant at membrane interfaces.

Transmembrane β-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 β-barrels can cause significant destabilization of these structural regions that require H-bonding, with proline additionally acting as a secondary structure breaker. Membrane protein β-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 Cterminal interface of the model transmembrane β-barrel PagP. We find that proline drastically destabilizes PagP by 7.0 kcal mol −1 with respect to wildtype PagP (F 161 → P 161 ). 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 β-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.