Backbone Hydrogen Bond Energies in Membrane Proteins Are Insensitive to Large Changes in Local Water Concentration

Abstract

A hallmark feature of biological lipid bilayer structure is a depth-dependent polarity gradient largely resulting from the change in water concentration over the angstrom length scale. This gradient is particularly steep as it crosses the membrane interfacial regions where the water concentration drops at least a million-fold along the direction of the bilayer normal. Although local water content is often assumed to be a major determinant of membrane protein stability, the effect of the water-induced polarity gradient upon backbone hydrogen bond strength has not been systematically investigated. We addressed this question by measuring the free energy change for a number of backbone hydrogen bonds in the transmembrane protein OmpW. These values were obtained at 33 backbone amides from hydrogen/deuterium fractionation factors by nuclear magnetic resonance spectroscopy. We surprisingly found that OmpW backbone hydrogen bond energies do not vary over a wide range of water concentrations that are characteristic of the solvation environment in the bilayer interfacial region. We validated the interpretation of our results by determining the hydrodynamic and solvation properties of our OmpW-micelle complex using analytical ultracentrifugation and molecular dynamics simulations. The magnitudes of the backbone hydrogen bond free energy changes in our study are comparable to those observed in water-soluble proteins, the H-segment of the leader peptidase helix used in the von Heijne and White biological scale experiments, and several interfacial peptides. Our results agree with those reported for the transmembrane α-helical portion of the amyloid precursor protein after the latter values were adjusted for kinetic isotope effects. Overall, our work suggests that backbone hydrogen bonds provide modest thermodynamic stability to membrane protein structures and that many amides are unaffected by dehydration within the bilayer.

Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria

“…This still requires the breaking of 5–7 hydrogen bonds, but some of this may be compensated by the kinked residues hydrogen-bonding to water within the lumen of the BamA barrel. Furthermore, the cost for OMPs to break hydrogen bonds in the membrane may be lower than previously assumed ( 372 ), and the outer leaflet of the OM may be more hydrated than a symmetric phospholipid bilayer. These effects could help stabilize the structure of BamA while effecting local changes in packing or stability of the membrane.…”

Local Bilayer Hydrophobicity Modulates Membrane Protein Stability

“…41 For all side chains, except Trp, , indicating that the translocon energetically mimics the interface . The deviation for Trp is hypothesized to be due to the energetic preference of Trp to exist in the bilayer interface 15,42 .…”

“…Although it was long expected that bbHB energies would also be strongly influenced by the steep water gradient intrinsic to bilayer interface, we recently observed bbHB energies to be constant throughout this region. 42 The experimental data here offer an opportunity to consider how the translocon insertion process relates to the underlying physical chemistry of the reaction. Our data provides evidence that translocon-mediated membrane protein folding energetically mimics the interface-to-bilayer transition for ∆" #$°.…”

“…Although it was long expected that bbHB energies would also be strongly influenced by the steep water gradient intrinsic to bilayer interface, we recently observed bbHB energies to be constant throughout this region. 42…”

Deep‐UV resonance Raman spectroscopy of hydrated and dehydrated model α‐helical transmembrane peptides in liposomes

“…Water hydrogen bonding (H‐bonding) to α‐helical transmembrane (TM) peptides serves multiple functions, [ 1 ] such as stabilization of helix conformation, [ 2–4 ] unfolding of helical peptides, [ 5 ] creating helical kinks, [ 6 ] or transportation of protons, ion, and water in channels through water wire. [ 7 ] Therefore, studying hydration along α‐helical TM peptide helps to better understand α‐helical peptide behavior and function.…”