Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers

ConspectusThe structures of membrane-bound polypeptides are intimately related to their functions and may change dramatically with the lipid environment. Circular dichroism (CD) is a rapid analytical method that requires relatively low amounts of material and no labeling. Conventional CD is routinely used to monitor the secondary structure of peptides and proteins <i>in solution</i>, for example, in the presence of ligands and other binding partners. In the case of membrane-active peptides and transmembrane proteins, these measurements can be applied to, and remain limited to, samples containing detergent micelles or small sonicated lipid vesicles. Such traditional CD analysis reveals only secondary structures. With the help of an oriented circular dichroism (OCD) setup, however, based on the preparation of <i>macroscopically oriented lipid bilayers</i>, it is possible to address the membrane alignment of a peptide in addition to its conformation. This approach has been mostly used for α-helical peptides so far, but other structural elements are conceivable as well. OCD analysis relies on Moffitt’s theory, which predicts that the electronic transition dipole moments of the backbone amide bonds in helical polypeptides are polarized either parallel or perpendicular to the helix axis. The interaction of the electric field vector of the circularly polarized light with these transitions results in an OCD spectrum of a membrane-bound α-helical peptide, which exhibits a characteristic line shape and reflects the angle between the helix axis and the bilayer normal. For parallel alignment of a peptide helix with respect to the membrane surface (S-state), the corresponding “fingerprint” CD band around 208 nm will exhibit maximum negative amplitude. If the helix changes its alignment via an obliquely tilted (T-state) to a fully inserted transmembrane orientation (I-state), the ellipticity at 208 nm decreases and the value approaches zero due to the decreased interactions between the field and the transition dipole.Compared to conventional CD, OCD data are not only collected in the biologically relevant environment of a highly hydrated planar lipid bilayer (whose composition can be varied at will), but in addition it provides information about the tilt angle of the polypeptide in the membrane. It is the method of choice for screening numerous different conditions, such as peptide concentration, lipid composition, membrane additives, pH, temperature, and sample hydration. All these factors have been found to affect the peptide alignment in membrane, while having little or no influence on conformation. In many cases, the observed realignment could be related to biological action, such as pore formation by antimicrobial and cell-penetrating peptides, or to binding events of transmembrane segments of integral membrane proteins. Likewise, any lipid-induced conversion from α-helix to β-sheeted conformation is readily picked up by OCD and has been interpreted in terms of protein instability or amyloid-formation

Scaling the Amphiphilic Character and Antimicrobial Activity of Gramicidin S by Dihydroxylation or Ketal Formation

The acid lability of aliphatic ketals, which often serve as protection groups for 1,2-diols, is influenced by their local structural environment. The acetonide of the protected amino acid <i>cis</i>-dihydroxyproline (Dyp) is a typical protecting group cleavable by traces of TFA. The tricyclic acetonide of the dipeptide d-HotTap is resistant to TFA and thus can serve as a bioorthogonal modification of bioactive peptides. With the aim of improving antimicrobial activity and hemolytic properties, we use these reactivity differences to scale the membrane affinity of the decapeptide Gramicidin S <i>cyclo</i>(d-Phe-Pro-Val-Orn-Leu-)₂ (<b>GS</b>). The <i>cis</i>-dihydroxylated amino acids are used to increase the polarity of GS or obversely decrease the polarity by stereoselective ketal formation with an aliphatic ketone. While Dyp (GS mimetic <b>15</b>) has only minimal influence on the biological properties of <b>GS</b>, d-HotTap at the position of d-Phe1-Pro2 eradicates the biological activity (GS mimetic <b>16</b>). The acid-stable ketals <b>17</b>–<b>19</b> are bioorthogonal modifications which reconstitute the biological activity of <b>GS</b>. We describe an improved synthesis of orthogonally protected Fmoc-Dyp-acetonide (<b>9</b>) and of several Fmoc-d-HotTap-ketals for solid-phase peptide synthesis

Transmembrane Polyproline Helix

The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold and demonstrate its transmembrane alignment in model lipid bilayers by means of solid-state ¹⁹F NMR. As result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding

Lipid Membrane Association of Myelin Proteins and Peptide Segments Studied by Oriented and Synchrotron Radiation Circular Dichroism Spectroscopy

Myelin-specific proteins are either integral or peripheral membrane proteins that, in complex with lipids, constitute a multilayered proteolipid membrane system, the myelin sheath. The myelin sheath surrounds the axons of nerves and enables rapid conduction of axonal impulses. Myelin proteins interact intimately with the lipid bilayer and play crucial roles in the assembly, function, and stability of the myelin sheath. Although myelin proteins have been investigated for decades, their structural properties upon membrane surface binding are still largely unknown. In this study, we have used simplified model systems consisting of synthetic peptides and membrane mimics, such as detergent micelles and/or lipid vesicles, to probe the conformation of peptides using synchrotron radiation circular dichroism spectroscopy (SRCD). Additionally, oriented circular dichroism spectroscopy (OCD) was employed to examine the orientation of myelin peptides in macroscopically aligned lipid bilayers. Various representative peptides from the myelin basic protein (MBP), P0, myelin/oligodencrocyte glycoprotein, and connexin32 (cx32) were studied. A helical peptide from the central immunodominant epitope of MBP showed a highly tilted orientation with respect to the membrane surface, whereas the N-terminal cytoplasmic segment of cx32 folded into a helical structure that was only slightly tilted. The folding of full-length myelin basic protein was, furthermore, studied in a bicelle environment. Our results provide information on the conformation and membrane alignment of important membrane-binding peptides in a membrane-mimicking environment, giving novel insights into the mechanisms of membrane binding and stacking by myelin proteins

Extending the Hydrophobic Mismatch Concept to Amphiphilic Membranolytic Peptides

A series of nine amphiphilic, pore-forming α-helical KIA peptides (KIAGKIA repeats) with lengths between 14 and 28 residues were studied by solid-state ¹⁵N NMR to determine their alignment in oriented lipid bilayers. In a 2:1 mixture of 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphatidylcholine (DMPC) with its corresponding 1-myristoyl-2-hydroxy-<i>sn</i>-glycero-3-phosphocholine (lyso-MPC), which has a highly positive spontaneous curvature, the helix tilt angle was found to vary steadily with peptide length. The shortest peptide was aligned transmembrane and upright, while the longer ones successively became tilted away from the membrane normal. This behavior is in agreement with the hydrophobic matching concept, conceived so far only for hydrophobic helices. In 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphatidylcholine, with a negative spontaneous curvature, all KIA peptides remained flat on the bilayer surface, while the cylindrical DMPC lipids permitted a slight tilt. Peptide insertion thus depends critically on the intrinsic lipid curvature, and helix orientation is then fine-tuned by membrane thickness. A refined toroidal pore model is proposed

Incorporation of <i>cis</i>- and <i>trans</i>-4,5-Difluoromethanoprolines into Polypeptides

Substituted prolines exert diverse effects on the backbone conformation of proteins. Novel difluoro-analogues were obtained by adding difluorocarbene to N-Boc-4,5-dehydroproline methyl ester, which gave the <i>trans</i>-adduct as the sole product with 71% yield. Upon cleavage of the N-protection group the free amino acid decomposed rapidly. Its incorporation into the proline-rich cell-penetrating “sweet arrow peptide” was thus accomplished using a dipeptide strategy. Two building blocks, containing either <i>cis</i>- or <i>trans</i>-4,5-difluoromethanoproline, were obtained by difluorocyclopropanation of the aminoacyl derivatives of 4,5-dehydroproline. The resulting dipeptides were stable under standard conditions of Fmoc solid phase peptide synthesis and, thus, suitable to study conformational effects

¹⁹F‑Labeling of Peptides Revealing Long-Range NMR Distances in Fluid Membranes

NMR distance measurements lie at the heart of structural biology. However, long-range distances could not yet be detected in liquid–crystalline biomembranes, because dipolar couplings are partially averaged by the intrinsic molecular mobility. Using conformationally constrained ¹⁹F-labeled amino acids as reporter groups, we could more than double the accessible interatomic distance range by combining a highly sensitive solid-state multipulse ¹⁹F-NMR scheme with a favorable sample geometry. Two rigid 4F-phenylglycine labels were placed into the helical antimicrobial peptide PGLa embedded in fluid oriented membrane samples. A modified Carr–Purcell–Meiboom–Gill sequence yielded an intramolecular distance of 6.6 Å for the labels spanning one helix turn, and 11.0 Å was obtained when the labels spanned two turns. This approach should now also allow the characterization of conformational changes in membrane-active peptides and of oligomeric assemblies in a biologically relevant lipid environment

Amphipathic helix model of the E^rns membrane anchor.

<p>2D flat projection of the 3D structure of the E^rns anchor (Lys167 – Ala227) assuming a continuous α-helical conformation. Positively charged amino acids are shown in dark blue (Arg, Lys), negatively charged ones in red (Asp, Glu), and hydrophobic amino acids are colored in yellow (Leu, Val, Ile, Met, Trp, Tyr, Phe, Ala, Cys). Polar amino acids are displayed in light blue (Thr, Asn, Ser, Gln, His), and the remaining ones (Gly, Pro) in green. The illustration was generated with the in-house software “Protein Origami” (Karlsruhe Institute of Technology, <a href="http://www.ibg.kit.edu/nmr/544.php" target="_blank">http://www.ibg.kit.edu/nmr/544.php</a>).</p

Structure of the Membrane Anchor of Pestivirus Glycoprotein E^rns, a Long Tilted Amphipathic Helix

<div><p>E^rns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E^rns membrane contact, processing and secretion.</p></div

Characterization of the Immersion Properties of the Peripheral Membrane Anchor of the FATC Domain of the Kinase “Target of Rapamycin” by NMR, Oriented CD Spectroscopy, and MD Simulations

The multidomain ser/thr kinase “target of rapamycin” (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438–2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn²⁺ in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30–60°/ ∼ 35–65° in neutral and ∼5–35°/∼0–30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein–protein and protein–membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins