Venkatesan Rajagopalan

Dynamic regulation of lipid–protein interactions ☆

We review the importance of helix motions for the function of several important categories of membrane proteins and for the properties of several model molecular systems. For voltage-gated potassium or sodium channels, sliding, tilting and/or rotational movements of the S4 helix accompanied by a swapping of cognate side-chain ion-pair interactions regulate the channel gating. In the seven-helix G protein-coupled receptors, exemplified by the rhodopsins, collective helix motions serve to activate the functional signaling. Peptides which initially associate with lipid-bilayer membrane surfaces may undergo dynamic transitions from surface-bound to tilted-transmembrane orientations, sometimes accompanied by changes in the molecularity, formation of a pore or, more generally, the activation of biological function. For single-span membrane proteins, such as the tyrosine kinases, an interplay between juxtamembrane and transmembrane domains is likely to be crucial for the regulation of dimer assembly that in turn is associated with the functional responses to external signals. Additionally, we note that experiments with designed single-span transmembrane helices offer fundamental insights into the molecular features that govern protein-lipid interactions. This article is part of a Special Issue entitled: Lipid-protein interactions.

Juxta-terminal Helix Unwinding as a Stabilizing Factor to Modulate the Dynamics of Transmembrane Helices.

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that can serve as anchors to aid the stabilization of a tilted transmembrane orientation. Yet, physical factors that govern the orientation or dynamic averaging of individual transmembrane helices are not well understood and have not been adequately explained. Using solid‐state 2H NMR spectroscopy to examine lipid bilayer‐incorporated model peptides of the GWALP23 (acetyl‐GGALW(LA)6LWLAGA‐amide) family, we observed substantial unwinding at the terminals of several tilted helices spanning the membranes of DLPC, DMPC, or DOPC lipid bilayers. The fraying of helix ends might be vital for defining the dynamics and orientations of transmembrane helices in lipid bilayer membranes.

Influence of glutamic acid residues and pH on the properties of transmembrane helices

Negatively charged side chains are important for the function of particular ion channels and certain other membrane proteins. To investigate the influence of single glutamic acid side chains on helices that span lipid-bilayer membranes, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as a favorable host peptide framework. We substituted individual Leu residues with Glu residues (L12E or L14E or L16E) and incorporated specific 2H-labeled alanine residues within the core helical region or near the ends of the sequence. Solid-state 2H NMR spectra reveal little change for the core labels in GWALP23-E12, -E14 and -E16 over a pH range of 4 to 12.5, with the spectra being broader for samples in DOPC compared to DLPC bilayers. The spectra for samples with deuterium labels near the helix ends on alanines 3 and 21 show modest pH-dependent changes in the extent of unwinding of the helix terminals in DLPC and DOPC bilayers. The combined results indicate minor overall responses of these transmembrane helices to changes in pH, with the most buried residue E12 showing no pH dependence. While the Glu residues E14 and E16 may have high pKa values in the lipid bilayer environment, it is also possible that a paucity of helix response is masking the pKa values. Interestingly, when E16 is present, spectral changes at high pH report significant local unwinding of the core helix. Our results are consistent with the expectation that buried carboxyl groups aggressively hold their protons and/or waters of hydration.

Helix Fraying May Stabilize Transmembrane Alpha Helices

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that may serve as anchors to aid the stabilization of a tilted transmembrane orientation. To further understand the influence of Tyr, Trp or Phe residues upon the properties of helical membrane proteins (see Biochemistry 53, 3637), we have investigated the possibility of partial unwinding near the ends of selected transmembrane helices. To this end, we have substituted positions 4 and 5 of GWALP23 with either two Phe residues or two Ala residues to generate F4,5GWALP23 (acetyl-GGAF4F5(LA)6LW19LAGA-ethanolamide) or A4,5GWALP23 (acetyl-GGAA4A5(LA)6LW19LAGA-ethanolamide). By incorporating specific 2H-Ala labels at A3 and A21, as well as within the (LA)6L core, we are able to compare the influence of interfacial residues on the integrity of the core helix and the extent of unwinding of the helix ends. Solid state 2H NMR spectra of macroscopically aligned bilayer samples indicate a well oriented, tilted core helix for the (LA)6L sequence of A4,5GWALP23 as well as F4,5GWALP23 in DLPC, DMPC and DOPC lipid bilayers. Furthermore, the spectra from deuterium labels on alanines 3 and 21 show substantial unwinding at the terminals for both F4,5GWALP23 and A4,5GWALP23. Further studies will address (a) the precise point of N-terminal unwinding of A4,5GWALP23 by use of 2H labels at A4 and A5 and (b) the possibility of unwinding of Y4,5GWALP23 and G4,5GWALP23 helices. The fraying of helix ends may be vital for the stability of the transmembrane helix orientation in lipid-bilayer membranes.

Investigating Possible Interactions between Ionizable Residues in Model Transmembrane Peptides

The study of charged residues in the hydrophobic interior of transmembrane proteins is crucial to the understanding of membrane proteins and their function. To this end, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide), a constructive low-dynamic model peptide, for investigations of single-residue influence on protein-lipid interactions (JBC 285, 31723). We have substituted a single Leu residue at position 12, within the hydrophobic core of the GWALP23 sequence, with either Arg (R12) or Glu (E12). Specific 2H-labeled Ala residues have been incorporated within the -R12 sequence for detection by solid-state 2H NMR. GWALP23-R12 remains charged in DOPC bilayers, even under strongly alkaline pH conditions, and displays multi-state behavior (JACS 132, 5803). To determine if the presence of an adjacent peptide with Glu at position 12 would influence the behavior of the -R12 peptide, we incorporate a mixture of labeled GWALP23-R12 and unlabeled -E12 peptides in DOPC bilayers. Preliminary results for oriented samples with both peptides suggest that -R12 remains multi-state in the presence of the -E12 peptide in DOPC bilayers at pH 6. By contrast, oriented samples of -R12 in DLPC bilayers show clearly defined quadrupolar splittings. In the presence of -E12, we observe small changes in the quadrupolar splittings, indicative of changes in orientation of the -R12 peptide. It is possible that peptide crowding within the bilayer may influence ionic interactions between the -E12 and -R12 peptides; therefore we are currently investigating the effect of varying the peptide/peptide and peptide/lipid ratios. Similar experiments with GW3,21ALP23-R12, with the Trp residues moved to the opposite face of the helix, also suggest interaction between -E12 and -R12 in both DLPC and DOPC bilayers at pH 6. Additionally, we are studying possible ion-pair interactions in a peptide with two ionizable residues, GWALP23-R12,E16.