Intrinsic Lipid Curvature and Bilayer Elasticity as Regulators of Channel Function: A Comparative Single-Molecule Study.

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the H lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels.

Membrane electrostatics sensed by tryptophan anchors in hydrophobic model peptides depends on non-aromatic interfacial amino acids: implications in hydrophobic mismatch.

WALPs are synthetic α-helical membrane-spanning peptides that constitute a well-studied system for exploring hydrophobic mismatch. These peptides represent a simplified consensus motif for transmembrane domains of intrinsic membrane proteins due to their hydrophobic core of alternating leucine and alanine flanked by membrane-anchoring aromatic tryptophan residues. Although the modulation of mismatch responses in WALPs by tryptophan anchors has been reported earlier, there have been limited attempts to utilize the intrinsic tryptophan fluorescence of this class of peptides in mismatch sensors.

Illuminating Disorder Induced by Glu in a Stable Arg-Anchored Transmembrane Helix.

Membrane proteins are vital for biological function and are complex to study. Even in model peptide-lipid systems, the combined influence or interaction of pairs of chemical groups still is not well understood. Disordered proteins, whether in solution or near lipid membranes, are an emerging paradigm for the initiation and control of biological function.

Lipid-Dependent Titration of Glutamic Acid at a Bilayer Membrane Interface.

The ionization properties of protein side chains in lipid-bilayer membranes will differ from the canonical values of side chains exposed to an aqueous solution. While the propensities of positively charged side chains of His, Lys, and Arg to release a proton in lipid membranes have been rather well characterized, the propensity for a negatively charged Glu side chain to receive a proton and achieve the neutral state in a bilayer membrane has been less well characterized. Indeed, the ionization of the glutamic acid side chain has been predicted to depend on its depth of burial in a lipid membrane but has been difficult to verify experimentally.

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-H, acetyl-GGALWLAHALALALALALWLAGA-amide, the deuterium NMR spectra of H-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.

Flanking aromatic residue competition influences transmembrane peptide helix dynamics.

To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW ALP23 (acetyl-GGAW A(LA) LAW AGA-amide) were examined using deuterium NMR spectroscopy. It was found that L GW ALP23, a sequence isomer of the low to moderately dynamic GW ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F GW ALP23 restores a low level of dynamic averaging, similar to that of the F GW ALP23 helix.

Influence of interfacial tryptophan residues on an arginine-flanked transmembrane helix.

The transmembrane helices of membrane proteins often are flanked by interfacial charged or aromatic residues that potentially help to anchor the membrane-spanning protein. For isolated single-span helices, the interfacial residues may be especially important for stabilizing particular tilted transmembrane orientations. The peptide RWALP23 (acetyl-GRALW(LA)LWLARA-amide) has been employed to investigate the interplay between interfacial arginines and tryptophans.

Breaking the Backbone: Central Arginine Residues Induce Membrane Exit and Helix Distortions within a Dynamic Membrane Peptide.

Transmembrane domains of membrane proteins sometimes contain conserved charged or ionizable residues which may be essential for protein function and regulation. This work examines the molecular interactions of single Arg residues within a highly dynamic transmembrane peptide helix. To this end, we have modified the GWALP23 (acetyl-GGAW(AL)AWAGA-amide) model peptide framework to incorporate Arg residues near the center of the peptide.

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-GGALW LAL LALALAL ALW LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains.

Antidepressants are modifiers of lipid bilayer properties.

The two major classes of antidepressants, tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), inhibit neurotransmitter reuptake at synapses. They also have off-target effects on proteins other than neurotransmitter transporters, which may contribute to both desired changes in brain function and the development of side effects. Many proteins modulated by antidepressants are bilayer spanning and coupled to the bilayer through hydrophobic interactions such that the conformational changes underlying their function will perturb the surrounding lipid bilayer, with an energetic cost (Δ ) that varies with changes in bilayer properties.

Transmembrane Helix Integrity versus Fraying To Expose Hydrogen Bonds at a Membrane-Water Interface.

Transmembrane helices dominate the landscape for many membrane proteins. Often flanked by interfacial aromatic residues, these transmembrane helices also contain loops and interhelix segments, which could help in stabilizing a transmembrane orientation. Using H nuclear magnetic resonance spectroscopy to monitor bilayer-incorporated model GWALP23 family peptides, we address systematically the issue of helix fraying in relation to the dynamics and orientation of highly similar individual transmembrane helices.

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors.

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans.

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.

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles.

Helix formation and stability in membranes.

In this article we review current understanding of basic principles for the folding of membrane proteins, focusing on the more abundant alpha-helical class. Membrane proteins, vital to many biological functions and implicated in numerous diseases, fold into their active conformations in the complex environment of the cell bilayer membrane. While many membrane proteins rely on the translocon and chaperone proteins to fold correctly, others can achieve their functional form in the absence of any translation apparatus or other aides.

Exchange of Gramicidin between Lipid Bilayers: Implications for the Mechanism of Channel Formation.

The canonical mechanism of gramicidin (gA) channel formation is transmembrane dimerization of nonconducting subunits that reside in opposite bilayer leaflets. The channels do not open and close; they appear and disappear due to subunit association and dissociation. Many different types of experiments support this monomer ↔ dimer mechanism.

Characterizing Residue-Bilayer Interactions Using Gramicidin A as a Scaffold and Tryptophan Substitutions as Probes.

Previous experiments have shown that the lifetime of a gramicidin A dimer channel (which forms from two nonconducting monomers) in a lipid bilayer is modulated by mutations of the tryptophan (Trp) residues at the bilayer-water interface. We explore this further using extensive molecular dynamics simulations of various gA dimer and monomer mutants at the Trp positions in phosphatidylcholine bilayers with different tail lengths. gA interactions with the surrounding bilayer are strongly modulated by mutating these Trp residues.

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-GGALWLALALALALALALWLAGA-amide) as a favorable host peptide framework. We substituted individual Leu residues with Glu residues (L12E or L14E or L16E) and incorporated specific H-labeled alanine residues within the core helical region or near the ends of the sequence.

Lipid bilayer thickness determines cholesterol's location in model membranes.

Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature.

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).

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).

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 (2) H NMR spectroscopy to examine lipid bilayer-incorporated model peptides of the GWALP23 (acetyl-GGALW(LA)6 LWLAGA-amide) family, we observed substantial unwinding at the terminals of several tilted helices spanning the membranes of DLPC, DMPC, or DOPC lipid bilayers.

A general mechanism for drug promiscuity: Studies with amiodarone and other antiarrhythmics.

Amiodarone is a widely prescribed antiarrhythmic drug used to treat the most prevalent type of arrhythmia, atrial fibrillation (AF). At therapeutic concentrations, amiodarone alters the function of many diverse membrane proteins, which results in complex therapeutic and toxicity profiles. Other antiarrhythmics, such as dronedarone, similarly alter the function of multiple membrane proteins, suggesting that a multipronged mechanism may be beneficial for treating AF, but raising questions about how these antiarrhythmics regulate a diverse range of membrane proteins at similar concentrations.

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.