Membrane Depth-Dependent Energetic Contribution of the Tryptophan Side Chain to the Stability of Integral Membrane Proteins

Abstract

Lipid solvation provides the primary driving force for the insertion and folding of integral membrane proteins. Although the structure of the lipid bilayer is often simplified as a central hydrophobic core sandwiched between two hydrophilic interfacial regions, the complexity of the liquid-crystalline bilayer structure and the gradient of water molecules across the bilayer finetune the energetic contributions of individual amino acid residues to the stability of membrane proteins at different depths of the bilayer. The tryptophan side-chain is particularly interesting because despite its widely recognized role in anchoring membrane proteins in lipid bilayers, there is little consensus on its hydrophobicity among various experimentally determined hydrophobicity scales. Here we investigated how lipid-facing tryptophan residues located at different depths in the bilayer contribute to the stability of integral membrane proteins using the outer membrane protein A (OmpA) as a model. We replaced all lipid-contacting residues of the first transmembrane β-strand of OmpA with alanines and individually incorporated tryptophans in these positions along the strand. By measuring the thermodynamic stability of these proteins, we found that OmpA is slightly more stable when tryptophans are placed in the center of the bilayer and that it is somewhat destabilized as tryptophans approach the interfacial region. However, this trend may be partially reversed when a moderate concentration of urea rather than water is taken as the reference state. The measured stability profiles are driven by similar profiles of the m-value, a parameter that reflects the shielding of hydrophobic surface area from water. Our results indicate that knowledge of the free energy level of the protein’s unfolded reference state is important for quantitatively assessing the stability of membrane proteins, which may explain differences in observed profiles between in vivo and in vitro scales.

Aromatic Side Chain Water-to-Lipid Transfer Free Energies Show a Depth Dependence across the Membrane Normal

“…In contrast to an early study, 22 our measurements indicate a thermodynamic enhancement for Trp and Tyr at the membrane interface as compared to the bilayer center. Our work shows that the depth-dependent free energy changes are well described by the concentration of polar atoms present in the membrane polarity gradient.…”

Differential Contribution of Tryptophans to the Folding and Stability of the Attachment Invasion Locus Transmembrane β-Barrel from Yersinia pestis

“…A recent study that estimated the influence of lipid solvation of Trp residues of E. coli OmpA found greater β-barrel stabilization when the indole is buried, i.e., is highly lipid solvated (buried indole)36. A complementary report investigating multi-pass TM helices provides contrasting evidence, wherein preferential interfacial Trp localization and a noticeable affinity of Phe for the lipid core were obtained52.…”

Control of human VDAC-2 scaffold dynamics by interfacial tryptophans is position specific

“…The diverse interaction network involving tryptophan allows this residue to serve as a strong anchoring point for membrane proteins. Recent experimental evidence points to a contextual contribution of the indole ring to the overall membrane protein stability [8]. In less polar environments, the contribution of tryptophan is highest at the lipid hydrophobic core, while in environments that resemble cellular conditions, the greatest stabilizing contribution for tryptophan was seen at the interface [8].…”

“…Recent experimental evidence points to a contextual contribution of the indole ring to the overall membrane protein stability [8]. In less polar environments, the contribution of tryptophan is highest at the lipid hydrophobic core, while in environments that resemble cellular conditions, the greatest stabilizing contribution for tryptophan was seen at the interface [8]. An overall stabilizing effect of tryptophans is, however, anticipated, depending upon its positioning according to membrane depth [9].…”