Stephan L. Grage

Temperature-dependent transmembrane insertion of the amphiphilic peptide PGLa in lipid bilayers observed by solid state 19F NMR spectroscopy.

The alignment of the antimicrobial peptide PGLa in a lipid bilayer was characterized by solid state 19F NMR on selectively CF3-labeled peptides in oriented samples. Previous studies in liquid crystalline model membranes had shown that the amphiphilic α-helix of PGLa adopts a surface-aligned S-state or a tilted T-state, depending on the peptide/lipid ratio. Only in the presence of the synergistic partner peptide magainin 2 had PGLa been found insert into the bilayer in a transmembrane I-state orientation. Here, we have characterized the peptide alignment but as a function of temperature and lipid phase state. At temperatures below the lipid chain melting transition, PGLa is now seen to be able to insert on its own, with its helical axis nearly parallel to the bilayer normal (tilt angle of ∼170°), forming the I-state. Above the lipid phase transition, PGLa is aligned in the known T-state (tilt angle of ∼130°), but it is found flip into the S-state at more elevated temperatures (tilt angle of ∼96°). This way...

Bilayer-Mediated Clustering and Functional Interaction of MscL Channels

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

Solid-State 19F-NMR Analysis of 19F-Labeled Tryptophan in Gramicidin A in Oriented Membranes

The response of membrane-associated peptides toward the lipid environment or other binding partners can be monitored by solid-state NMR of suitably labeled side chains. Tryptophan is a prominent amino acid in transmembrane helices, and its (19)F-labeled analogues are generally biocompatible and cause little structural perturbation. Hence, we use 5F-Trp as a highly sensitive NMR probe to monitor the conformation and dynamics of the indole ring. To establish this (19)F-NMR strategy, gramicidin A was labeled with 5F-Trp in position 13 or 15, whose chi(1)/chi(2) torsion angles are known from previous (2)H-NMR studies. First, the alignment of the (19)F chemical shift anisotropy tensor within the membrane was deduced by lineshape analysis of oriented samples. Next, the three principal axes of the (19)F chemical shift anisotropy tensor were assigned within the molecular frame of the indole ring. Finally, determination of chi(1)/chi(2) for 5F-Trp in the lipid gel phase showed that the side chain alignment differs by up to 20 degrees from its known conformation in the liquid crystalline state. The sensitivity gain of (19)F-NMR and the reduction in the amount of material was at least 10-fold compared with previous (2)H-NMR studies on the same system and 100-fold compared with (15)N-NMR.

Hydrophobic Matching Controls the Tilt and Stability of the Dimeric Platelet-derived Growth Factor Receptor (PDGFR) β Transmembrane Segment

Background: Dimerization regulates activation of PDGF receptor in signal transduction. Results: The transmembrane segment of PDGFR forms a left-handed helical dimer, which becomes more tilted and less stable in model membranes with decreasing lipid acyl chain lengths. Conclusion: The membrane thickness controls the ability of the transmembrane segments to dimerize. Significance: Receptor dimerization and activation in vivo may require relocation to thick lipid rafts. The platelet-derived growth factor receptor β is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor β transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo.

Solid state 19F NMR parameters of fluorine-labeled amino acids. Part II: Aliphatic substituents

A representative set of amino acids with aliphatic 19F-labels has been characterized here, following up our previous compilation of NMR parameters for single 19F-substituents on aromatic side chains. Their isotropic chemical shifts, chemical shift tensor parameters, intra-molecular 19F dipole-dipole couplings and temperature-dependent T1 and T2 relaxation times were determined by solid state NMR on twelve polycrystalline amino acid samples, and the corresponding isotropic 19F chemical shifts and scalar couplings were obtained in solution. Of particular interest are amino acids carrying a trifluoromethyl-group, because not only the 19F chemical shift but also the intra-CF3 homonuclear dipolar coupling can be used for structural studies of 19F-labeled peptides and proteins. The CF3-groups are further compared with CH2F-, CD2F-, and CD3-groups, using both 19F and 2H NMR to describe their motional behavior and to examine the respective linebroadening effects of the protonated and deuterated neighbors. We have also characterized two unnatural amino acids in which a CF3-label is rigidly connected to the backbone by a phenyl or bicyclopentyl moiety, and which are particularly well suited for structure analysis of membrane-bound polypeptides. The 19F NMR parameters of the polycrystalline amino acids are compared with data from the correspondingly labeled side chains in synthetic peptides.

Hydration of DMPC and DPPC at 4°C produces a novel subgel phase with convex–concave bilayer curvatures

Abstract Hydration of dimyristoyl- and dipalmitoylphosphatidylcholines at 4°C results in the formation of a characteristic subgel phase designated Pcc. Examination of the phase by freeze-fracture electron microscopy shows convex–concave deformations of the planar bilayer which are of two types. A smaller type with a radius of curvature of about 20 nm predominates in DMPC, and a larger type with about 70 nm radii of curvatures dominates in DPPC. The Pcc phase can also be formed in samples hydrated at temperatures above the main phase transition if the dispersion is frozen slowly and subsequently incubated at 4°C for several days. The subgel Pcc phase was distinguished from the subgel Lc phase by the temperature of transition, packing of the acyl chains on the basis of wide-angle X-ray diffraction, and 2H-NMR spectra characteristic of a ‘solid-ordered’ phase. Vibrational spectra of the carbonyl and phosphate regions are consistent with a partially reduced hydration state. The origin of the convex–concave bilayer deformation is believed to result from constraints imposed by limiting hydration of the headgroup and a frustration arising from the spontaneous curvature of both monolayers.

Click chemistry produces hyper-cross-linked polymers with tetrahedral cores

Methane and adamantane based hyper-cross-linked polymers have been prepared by click chemistry reacting the corresponding tetraalkynes with 1,4-diazidobenzene. The adamantane based HCP proved to be very efficient for CO2 capture at low pressures.

Dynamic Transitions of Membrane-Active Peptides

Membrane-active peptides or protein segments play an important role in many biological processes at the cellular interface to the environment. They are involved, e.g., in cellular fusion or host defense, where they can cause not only merging but also the destabilization of cell membranes. Many factors determine how these typically amphipathic peptides interact with the lipid bilayer. For example, the peptide orientation in the membrane determines which parts of the peptide are exposed to the hydrophobic bilayer interior or to the polar lipid/water interface. As another example, oligomerization is required for many activities such as pore formation. Peptides have been often classified according to a single characteristic mode of interaction with the bilayer, but over the years a more versatile picture has emerged. It appears that any single peptide can adopt several different alignments and/or oligomeric states in response to changes in the environment. For instance, many antimicrobial peptides adopt a surface-parallel alignment at low concentration, but they tilt obliquely into or even fully insert transmembrane into the bilayer above a critical peptide-to-lipid ratio, often in the form of oligomeric pores. Similar changes in peptide orientation or oligomeric state have been observed as a function of, e.g., temperature, lipid composition, pH, or induced by a synergistic partner peptide. Such transitions between peptide states can be regarded as the result of a re-adjustment in the balance between peptide-peptide and peptide-lipid interactions, as the environment conditions are changed. Though often studied in model membrane systems, such rich variety of peptide states is even more likely to occur in native biomembranes with their diverse compositions and physicochemical properties. The ability to undergo transitions between different states thus plays a fundamental role for the biological activities of membrane-active peptides.

Membrane Thickening by the Antimicrobial Peptide PGLa

Using x-ray diffraction, solid-state 2H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.

Labile or Stable: Can Homoleptic and Heteroleptic PyrPHOS–Copper Complexes Be Processed from Solution?

Luminescent Cu(I) complexes are interesting candidates as dopants in organic light-emitting diodes (OLEDs). However, open questions remain regarding the stability of such complexes in solution and therefore their suitability for solution processing. Since the emission behavior of Cu(I) emitters often drastically differs between bulk and thin film samples, it cannot be excluded that changes such as partial decomposition or formation of alternative emitting compounds upon processing are responsible. In this study, we present three particularly interesting candidates of the recently established copper-halide-(diphenylphosphino)pyridine derivatives (PyrPHOS) family that do not show such changes. We compare single crystals, amorphous bulk samples, and neat thin films in order to verify whether the material remains stable upon processing. Solid-state nuclear magnetic resonance (MAS (31)P NMR) was used to investigate the electronic environment of the phosphorus atoms, and X-ray absorption spectroscopy at the Cu K edge provides insight into the local electronic and geometrical environment of the copper(I) metal centers of the samples. Our results suggest that--unlike other copper(I) complexes--the copper-halide-PyrPHOS clusters are significantly more stable upon processing and retain their initial structure upon quick precipitation as well as thin film processing.