Jörg Nikolaus

Membrane-Mediated Induction and Sorting of K-Ras Microdomain Signaling Platforms

The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (l(d)) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the l(d) phase and concentrate at the l(d)/l(o) phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.

The Lipid Modifications of Ras that Sense Membrane Environments and Induce Local Enrichment

The transduction of an external stimulus from the outside of a cell into its nucleus is one of the most important mechanisms for the regulation of numerous biological processes. External signals activate receptors that transmit the information across the membrane, where it is transducted by a set of proteins that activate ion channels, phosphokinases, or other downstream effectors. GTP binding proteins that pick up the signal at the receptor, such as heterotrimeric G proteins or Ras, are membrane-associated by post-translationally acquired lipid modifications. These lipid chains provide the hydrophobic free energy for membrane association and their lack releases the proteins to the cytosol, rendering them inactive. Thus, through membrane binding, Ras increases its effective concentration to optimize the interaction both with the receptor and downstream effectors. Ras is an important molecular switch that regulates cell proliferation, differentiation, and growth. The highly specific membrane binding of Ras can be appreciated by comparing the members of the Ras family: Two lipid modifications are required for N-Ras and K-Ras4A, whereas H-Ras carries three lipid chains. In contrast, K-Ras4B requires the concerted action of one lipid chain and favorable electrostatics for membrane binding. Although inserted into the membrane, the lipid modifications experience a high degree of motional freedom that is also transmitted to the adjacent polypeptide chain. Although the highly homologous Ras proteins interact with the same effectors in vitro, they produce distinctly different output signals in vivo, which suggests that these differences are imparted by the lipid-modified C termini of the proteins, where the homology is very low. Moreover, depending on the nucleotide binding state, the localization of Ras in liquid-crystalline or raft domains of the membrane appears to be regulated. Only active H-Ras*GTP interacts with the respective set of effectors; the non-activated form, H-Ras*GDP, is constrained to rafts, where the signal is not further transmitted. An alternative model suggests that the difference in signaling of the Ras isoforms is imparted from the altered access and residence time in a specific compartment. 10] This model suggests that interactions of Ras and its lipid modifications with rafts or fluid membrane domains determines the membrane localization and the biological function of the molecule, which is investigated herein. H NMR is a useful tool for the investigation of lipid rafts. It is applicable to each component of a lipid mixture, and only requires the synthesis of the relevant molecule with a deuterated chain. First, we investigated the adaptation of the lipid modifications of a N-Ras heptapeptide, which was hexadecylated at Cys181 and Cys186, to the membrane thickness. Four different membranes composed of lipids with varying hydrocarbon chains were chosen to constitute the host membrane. Membrane thicknesses studied by H NMR varied from 21.0 (DLPC) to 38.8 (DPPC/cholesterol 10:6, Table 1). The high cholesterol content leads to condensation of the lipids, which increases their length and abolishes the phase transitions of DPPC such that all lipid mixtures could be studied at 30 8C.

Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.

An Amphiphilic Perylene Imido Diester for Selective Cellular Imaging

A new amphiphilic membrane marker based on a water-soluble dendritic polyglycerol perylene imido dialkylester has been designed, synthesized, and its optical properties characterized. In water it forms fluorescently quenched micellar self-aggregates, but when incorporated into a lipophilic environment, it monomerizes, and the highly fluorescent properties of the perylene core are recovered. These properties make it an ideal candidate for the imaging of artificial and cellular membranes as demonstrated by biophysical studies.

Cholesterol’s Aliphatic Side Chain Modulates Membrane Properties†

The influence of cholesterols alkyl side chain on membrane properties was studied using a series of synthetic cholesterol derivatives without a side chain or with a branched side chain consisting of 5 to 14 carbon atoms. Cholesterols side chain is crucial for all membrane properties investigated and therefore essential for the membrane properties of eukaryotic cells.

Direct visualization of large and protein-free hemifusion diaphragms.

Fusion of cellular membranes is a ubiquitous biological process requiring remodeling of two phospholipid bilayers. We believe it is very likely that merging of membranes proceeds via similar sequential intermediates. Contacting membranes form a stalk between the proximal leaflets that expands radially into an hemifusion diaphragm (HD) and subsequently open to a fusion pore. Although considered to be a key intermediate in fusion, direct experimental verification of this structure is difficult due to its transient nature. Using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (GUVs) containing phosphatidylserine and fluorescent virus derived transmembrane peptides or membrane proteins in the presence of divalent cations. Time-resolved imaging revealed that fusion was preceded by displacement of peptides and fluorescent lipid analogs from the GUV-GUV adhesion region. A detailed analysis of this area being several mum in size revealed that peptides were completely sequestered as expected for an HD. Lateral distribution of lipid analogs was consistent with formation of an HD but not with the presence of two adherent bilayers. Formation and size of the HD were dependent on lipid composition and peptide concentration.

Secondary structure and distribution of fusogenic LV-peptides in lipid membranes

LV-peptides were designed as membrane-spanning low-complexity model structures that mimic fusion protein transmembrane domains. These peptides harbor a hydrophobic core sequence that consists of helix-promoting and helix-destabilizing residues at different ratios. Previously, the fusogenicity of these peptides has been shown to increase with the conformational flexibility of their hydrophobic cores as determined in isotropic solution. Here, we examined the secondary structure, orientation, and distribution of LV-peptides in membranes. Our results reveal that the peptides are homogeneously distributed within the membranes of giant unilamellar liposomes and capable of fusing them. Increasing the valine content of the core up to the level of the β-branched residue content of SNARE TMDs (∼50%) enhances fusogenicity while maintaining a largely α-helical structure in liposomal membranes. A further increase in valine content or introduction of a glycine/proline pair favors β-sheet formation. In planar bilayers, the α-helices adopt oblique angles relative to the bilayer normal and the ratio of α-helix to β-sheet responds more sensitively to valine content. We propose that the fusogenic conformation of LV-peptides is likely to correspond to a membrane-spanning α-helix. β-Sheet formation in membranes may be considered a side-reaction whose extent reflects conformational flexibility of the core.

DBD dyes as fluorescent probes for sensing lipophilic environments

Small fluorescent organic molecules based on [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) could be used as probes for lipophillic microenvironments in aqueous solutions by indicating the critical micelles concentration of detergents and staining cell organelles. Their fluorescence lifetime decreases drastically by the amount of water in their direct environment. Therefore they are potential probes for fluorescence lifetime imaging microscopy (FLIM).

New molecular rods--characterization of their interaction with membranes.

Molecular rods are synthetical molecules consisting of a hydrophobic backbone which are functionalized with varying terminal groups. Here, we report on the interaction of a recently described new class of molecular rods with lipid and biological membranes. In order to characterize this interaction, different fluorescently labeled rods were synthesized allowing for the application of fluorescence spectroscopy and microscopy based approaches. Our data show that the rods are incorporated into membranes with a perpendicular orientation to the membrane surface and enrich preferentially in liquid-disordered lipid domains. These characteristics underline that rods can be applied as stable membrane-associated anchors for functionalizing membrane surfaces.

Molecular Rods with Oligospiroketal Backbones as Anchors in Biological Membranes

Getting stuck in: A hydrophobic molecular rod with terminal fluorescent moieties has been synthesized. The insertion of the rod into membranes was investigated and shown to incorporate efficiently into model and biological membranes (see picture; gray C, blue N, red O). Those rods can be used as stable membrane-associated anchors for functionalization of membrane surfaces.