Nicholas J. Gleason

Buried lysine, but not arginine, titrates and alters transmembrane helix tilt

The ionization states of individual amino acid residues of membrane proteins are difficult to decipher or assign directly in the lipid–bilayer membrane environment. We address this issue for lysines and arginines in designed transmembrane helices. For lysines (but not arginines) at two locations within dioleoyl-phosphatidylcholine bilayer membranes, we measure pKa values below 7.0. We find that buried charged lysine, in fashion similar to arginine, will modulate helix orientation to maximize its own access to the aqueous interface or, if occluded by aromatic rings, may cause a transmembrane helix to exit the lipid bilayer. Interestingly, the influence of neutral lysine (vis-à-vis leucine) upon helix orientation also depends upon its aqueous access. Our results suggest that changes in the ionization states of particular residues will regulate membrane protein function and furthermore illustrate the subtle complexity of ionization behavior with respect to the detailed lipid and protein environment.

Interactions of drugs and amphiphiles with membranes: modulation of lipid bilayer elastic properties by changes in acyl chain unsaturation and protonation

Poly-unsaturated fatty acids (PUFAs) alter the function of many membrane proteins, whereas monounsatured fatty acids generally are inert. We previously showed that docosahexaenoic acid (DHA) at pH 7 decreases the bilayer stiffness, consistent with an amphiphile-induced increase in elasticity, but not with a negative change in curvature; oleic acid (OA) was inert (Bruno, Koeppe and Andersen, Proc. Natl. Acad. Sci., 2007, 104, 9638-9643). To further explore how PUFAs and other amphiphiles may alter lipid bilayer properties, and thus membrane protein function, we examined how changes in acyl chain unsaturation and head group charge and size alter bilayer properties, as sensed by bilayer-spanning gramicidin A (gA) channels of different lengths. Compared to DHA, the neutral DHA-methyl ester has reduced effects on bilayer properties and 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine (PDPC) forms bilayers that are softer than dioleoylphosphatidylcholine (DOPC). The changes in channel function are larger for the short gA channels, indicating that changes in elasticity dominate over changes in curvature. We altered the fatty acid protonation by titration: docosahexaenoic acid (DHA) is more potent at pH 9 (relative to pH 7) and is inert at pH 4; OA, which was inert at pH 7, becomes a potent modifier of bilayer properties at pH 9. At both pH 7 and 9, DHA and OA produced larger changes in the lifetimes of the short gA channels, demonstrating that they increase lipid bilayer elasticity when deprotonated--though OA promotes the formation of inverted hexagonal phases at pH 7. The positively charged oleylamine (OAm), which has a small head-group and therefore should be a negative curvature promoter, inhibited gA channel function with similar reductions in the lifetimes of the short and long gA channels, indicating a curvature-dominated effect. Monitoring the single-channel conductance, we find that the negatively charged fatty acids increase the conductance by increasing the local negative charge around the channel, whereas the positively charged OAm has no effect. These results suggest that deprotonated fatty acids increase bilayer elasticity by reversibly adsorbing at the bilayer/solution interface.

Comparisons of Interfacial Phe, Tyr, and Trp Residues as Determinants of Orientation and Dynamics for GWALP Transmembrane Peptides

Aromatic amino acids often flank the transmembrane alpha helices of integral membrane proteins. By favoring locations within the membrane–water interface of the lipid bilayer, aromatic residues Trp, Tyr, and sometimes Phe may serve as anchors to help stabilize a transmembrane orientation. In this work, we compare the influence of interfacial Trp, Tyr, or Phe residues upon the properties of tilted helical transmembrane peptides. For such comparisons, it has been critical to start with no more than one interfacial aromatic residue near each end of a transmembrane helix, for example, that of GWALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide). To this end, we have employed 2H-labeled alanines and solid-state NMR spectroscopy to investigate the consequences of moving or replacing W5 or W19 in GWALP23 with selected Tyr, Phe, or Trp residues at the same or proximate locations. We find that GWALP23 peptides having F5, Y5, or W5 exhibit essentially the same average tilt and similar dynamics in bilayer membranes of 1,2-dilauroylphosphatidylcholine (DLPC) or 1,2-dioleoylphosphatidylcholine (DOPC). When double Tyr anchors are present, in Y4,5GWALP23 the NMR observables are markedly more subject to dynamic averaging and at the same time are less responsive to the bilayer thickness. Decreased dynamics are nevertheless observed when ring hydrogen bonding is removed, such that F4,5GWALP23 exhibits a similar extent of low dynamic averaging as GWALP23 itself. When F5 is the sole aromatic group in the N-interfacial region, the dynamic averaging is (only) slightly more extensive than with W5, Y5, or Y4 alone or with F4,5, yet it is much less than that observed for Y4,5GWALP23. Interestingly, moving Y5 to Y4 or W19 to W18, while retaining only one hydrogen-bond-capable aromatic ring at each interface, maintains the low level of dynamic averaging but alters the helix azimuthal rotation. The rotation change is about 40° for Y4 regardless of whether the host lipid bilayer is DLPC or DOPC. The rotational change (Δρ) is more dramatic and more complex when W19 is moved to W18, as Δρ is about +90° in DLPC but about −60° in DOPC. Possible reasons for this curious lipid-dependent helix rotation could include not only the separation distances between flanking aromatic or hydrophobic residues but also the absolute location of the W19 indole ring. For the more usual cases, when the helix azimuthal rotation shows little dependence on the host bilayer identity, excepting W18GWALP23, the transmembrane helices adapt to different lipids primarily by changing the magnitude of their tilt. We conclude that, in the absence of other functional groups, interfacial aromatic residues determine the preferred orientations and dynamics of membrane-spanning peptides. The results furthermore suggest possibilities for rotational and dynamic control of membrane protein function.