Abstract
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.
Highlights
The ability of histidine to participate in a wide range of stabilizing polar interactions preferentially populates this residue in functionally important sites of proteins
When we assess the energetic contribution of each residue by considering total free energy ΔΔG°U→N for the two-state mutants and the individual free energy values, ΔΔG°U→I or ΔΔG°I→N, for the three-state mutants, we find a heterogeneous distribution of residues with no specific hydrophobicity trend apparent in the analysis (Figure S5)
Our findings using PagP as our model protein are in good agreement with a previous observation that hydrophobicity, and not aromaticity, is the driving factor behind stabilization at the C-terminal interface of outer membrane proteins (OMPs).[22]
Summary
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 is the most versatile amino acid, because of its unique structural characteristics and its ability to participate in a wide range of intermolecular interactions.[1,2] The His side chain is considered polar in nature, with the ability to attain a positive charge dictated by the pH of the environment. Membrane proteins themselves possess a unique architecture, with hydrophobic functional groups presented to the surrounding lipid environment and a polar surface interacting with the aqueous milieu.[7,9,10] Within membrane proteins, His is known to play a key role in promoting protein−lipid interactions, at the interface, by means of interaction between the positively charged imidazole side chain and the negatively charged headgroup of lipid moieties,[11−14] yet the frequency of occurrence of His in transmembrane helices is lower than those of Phe, Tyr, and Trp; the frequency is decreased further in bacterial outer membrane proteins (OMPs).[7] A reasonable assumption based on existing hydrophobicity scales[15−18] would be that His is thermodynamically less favored than the other aromatic residues. By performing free energy measurements under alkaline conditions (pH 9.5), we can alter the ionized state of the His side chain (pKa of 6.0) and render it as an aromatic residue as opposed to a polar molecule
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