Examination of pH dependency and orientation differences of membrane spanning alpha helices carrying a single or pair of buried histidine residues.

We have employed the peptide framework of GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide) to examine the orientation, dynamics and pH dependence of peptides having buried single or pairs of histidine residues. When residue L8 is substituted to yield GWALP23-H8, acetyl-GGALWLAH8ALALALALALWLAGA-amide, the deuterium NMR spectra of 2H-labeled core alanine residues reveal a helix that occupies a single transmembrane orientation in DLPC, or in DMPC at low pH, yet shows multiple states at higher pH or in bilayers of DOPC. Moreover, a single histidine at position 8 or 16 in the GWALP23 framework is sensitive to pH. Titration points are observed near pH 3.5 for the deprotonation of H8 in lipid bilayers of DLPC or DMPC, and for H16 in DOPC. When residues L8 and L16 both are substituted to yield GWALP23-H8,16, the 2H NMR spectra show, interestingly, no titration dependence from pH 2-8, yet bilayer thickness-dependent orientation differences. The helix with H8 and H16 is found to adopt a transmembrane orientation in thin bilayers of DLPC, a combination of transmembrane and surface orientations in DMPC, and then a complete transition to a surface bound orientation in the thicker DPoPC and DOPC lipid bilayers. In the surface orientations, alanine A7 no longer fits within the core helix. These results along with previous studies with different locations of histidine residues suggest that lipid hydrophobic thickness is a first determinant and pH a second determinant for the helical orientation, along with possible side-chain snorkeling, when the His residues are incorporated into the hydrophobic region of a lipid membrane-associated helix.

Influence of Lipid Saturation, Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes.

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW5 LAL8 LALALAL16 ALW19 LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific 2 H-labeled alanine residues within the central sequence for detection by solid-state 2 H NMR spectroscopy. The resulting pattern of [2 H]Ala quadrupolar splitting (Δνq ) magnitudes indicates the core helix for R8,16 GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R8,16 GWALP23 helix adopts primarily a surface orientation. The inclusion of 10-20 mol % cholesterol in DOPC bilayers drives more of the R8,16 GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues.

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW5,19ALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2H- and 15N-labeled residues. GW5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2H quadrupolar or 15N-1H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW5,19ALP23, the modified GW4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2H and 15N NMR signals confirm that the extent of dynamic averaging, particularly rotational "slippage" about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.

Inactivation of Mechanically Activated Piezo1 Ion Channels Is Determined by the C-Terminal Extracellular Domain and the Inner Pore Helix.

Piezo proteins form mechanically activated ion channels that are responsible for our sense of light touch, proprioception, and vascular blood flow. Upon activation by mechanical stimuli, Piezo channels rapidly inactivate in a voltage-dependent manner through an unknown mechanism. Inactivation of Piezo channels is physiologically important, as it modulates overall mechanical sensitivity, gives rise to frequency filtering of repetitive mechanical stimuli, and is itself the target of numerous human disease-related channelopathies that are not well understood mechanistically. Here, we identify the globular C-terminal extracellular domain as a structure that is sufficient to confer the time course of inactivation and a single positively charged lysine residue at the adjacent inner pore helix as being required for its voltage dependence. Our results are consistent with a mechanism for inactivation that is mediated through voltage-dependent conformations of the inner pore helix and allosteric coupling with the C-terminal extracellular domain.

Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices.

An essential component of mammalian cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn its constituents, including membrane proteins. Although sparse, polar amino acid residues are highly conserved in membrane proteins and play pivotal roles in determining specific structural and functional properties. To improve our understanding of particular polar residues in the membrane environment, we have examined two specific "guest" Arg residues within a well-defined and deuterium-labeled "host" framework provided by the transmembrane helical peptide GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). Solid-state 2H nuclear magnetic resonance (NMR) spectra from aligned bilayer membrane samples effectively report changes in the host helix properties because of the incorporation of the guest residues. The focus of this work is two-pronged. First, GWALP23-R14 was examined over a pH range of 2-13 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ester- or ether-linked bilayer membranes. Our findings indicate that the Arg guanidinium side chain remains charged over this entire range, in agreement with numerous molecular dynamics simulations. Second, GWALP23-R12 and GWALP23-R14 peptides were characterized in DOPC bilayers with varying cholesterol content. Our findings suggest that 10 or 20% cholesterol content has minimal impact on the orientation of the R14 peptide. Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for [2H]Ala residues in GWALP23-R14 change very little. Conversely, cholesterol appears to modulate the multistate behavior of GWALP23-R12 and to favor a major interfacial state for the helix, bound at the bilayer surface. These results indicate a conditional sensitivity of a complex multistate transmembrane Arg-containing peptide helix to the presence of cholesterol.

Ionization Properties of Histidine Residues in the Lipid Bilayer Membrane Environment.

We address the critically important ionization properties of histidine side chains of membrane proteins, when exposed directly to lipid acyl chains within lipid bilayer membranes. The problem is important for addressing general principles that may underlie membrane protein function. To this end, we have employed a favorable host peptide framework provided by GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-amide). We inserted His residues into position 12 or 14 of GWALP23 (replacing either Leu(12) or Leu(14)) and incorporated specific [(2)H]Ala labels within the helical core sequence. Solid-state (2)H NMR spectra report the folding and orientation of the core sequence, revealing marked differences in the histidine-containing transmembrane helix behavior between acidic and neutral pH conditions. At neutral pH, the GWALP23-H12 and GWALP23-H14 helices exhibit well defined tilted transmembrane orientations in dioleoylphosphatidylcholine (DOPC)and dilauroylphosphatidylcholine (DLPC) bilayer membranes. Under acidic conditions, when His(12) is protonated and charged, the GWALP23-H12 helix exhibits a major population that moves to the DOPC bilayer surface and a minor population that occupies multiple transmembrane states. The response to protonation of His(14) is an increase in helix tilt, but GWALP23-H14 remains in a transmembrane orientation. The results suggest pKa values of less than 3 for His(12) and about 3-5 for His(14) in DOPC membranes. In the thinner DLPC bilayers, with increased water access, the helices are less responsive to changes in pH. The combined results enable us to compare the ionization properties of lipid-exposed His, Lys, and Arg side chains in lipid bilayer membranes.

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.