Transmembrane Domain Peptide Research Articles

A CD36 transmembrane domain peptide interrupts CD36 interactions with membrane partners on macrophages and inhibits atherogenic functions

CD36 is a transmembrane glycoprotein receptor for oxidized low density lipoprotein (LDL) and other endogenous danger signals and promotes athero-thrombotic processes. CD36 has been shown to associate physically with other transmembrane proteins, including integrins, tetraspanins, and toll-like receptors, which modulate CD36-mediated cell signaling. The CD36 N-terminal transmembrane domain (nTMD) contains a GXXXG sequence motif that mediates protein-protein interactions in many membrane proteins. We thus hypothesized that the nTMD is involved in CD36 interactions with other membrane proteins. CD36 interactions with partner cell surface proteins on murine peritoneal macrophages were detected with an immunofluorescence-based proximity ligation cross linking assay (PLA) and confirmed by immunoprecipitation/immunoblot. Prior to performing these assays, cells were incubated with a synthetic 29 amino acid peptide containing the 22 amino acid of CD36 nTMD or a control peptide in which the glycine residues in GXXXG motif were replaced by valines. In functional experiments, macrophages were preincubated with peptides and then treated with oxLDL to assess LDL uptake, foam cell formation, ROS formation and cell migration. CD36 nTMD peptide treated cells compared to untreated or control peptide treated cells showed decreased CD36 surface associations with tetraspanin CD9 and ameliorated pathologically important CD36 mediated responses to oxLDL, including uptake of DiI-labeled oxLDL, foam cell formation, ROS generation, and inhibition of migration.

Ni2+ Catalyzed Cleavage of TrpLE-Fused Small Transmembrane Peptides.

In addition to a membrane anchor, the transmembrane domain (TMD) of single-pass transmembrane proteins (SPTMPs) recently has shown essential roles in the cross-membrane activity or receptor assembly/clustering. However, these small TMD peptides are generally hydrophobic and dynamic, difficult to be expressed and purified. Here, we have integrated the power of TrpLE fusion protein and a sequence-specific nickel-assisted cleavage (SNAC)-tag to produce small TMD peptides in a highly efficient way under mild conditions, which uses Ni2+ as the cleavage reagent, avoiding the usage of toxic cyanogen bromide (CNBr). Furthermore, this method simplifies the downstream protein purification and reconstitution. Two representative TMDs, including the Spike-TMD from severe acute respiratory syndrome coronavirus 2 (SARS2), were successfully produced with high-quality nuclear magnetic resonance (NMR) spectra. Therefore, our study provides a more efficient and practical approach for general structural characterization of the small TM proteins.

Design and Generation of Self-Assembling Peptide Virus-like Particles with Intrinsic GPCR Inhibitory Activity.

Synthetic analogs of the second transmembrane domain (TM) containing a portion of the extracellular loop 1 of G-protein-coupled receptors (GPCR) can serve as biased antagonists of the corresponding receptor. Analogs with negative charges added to the extracellular end self-assemble into round structures. Addition of polyethylene glycol chains of defined length to the C-terminus of the peptides prevents super aggregation and results in highly uniform particles that can fuse with cell membranes spontaneously. Added PEG chains slow down cell fusion, while attachment of receptor ligands to the surface of particles results in receptor-mediated membrane fusion and cell-selective delivery. Critical assembly concentration of TM peptide particles is in the nanomolar range and thus requires nontraditional methods of determination. In this chapter, we outline sequence selection and design of self-assembling GPCR antagonists, methods of the preparation of the nanoparticles, and biophysical methods of particle characterization. The protocols allow for straightforward rational design, generation, and characterization of self-assembling GPCR antagonists for a variety of applications.

Curvature effect and stabilize ruptured membrane of BAX derived peptide studied by molecular dynamics simulations

BAX protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. The C-terminal transmembrane domain (TMD) of BAX is supposed to act a membrane anchor when BAX is activated leading to programmed cell death. Previous studies indicate that the C-terminal transmembrane domain of BAX mediates membrane disruption and pore formation, however, the mechanism of the membrane disruption and pore-forming capability of BAX C-terminal transmembrane domain still unclear. Here, we performed all-atom (AA) molecular dynamics simulations to study the membrane effect of TMD peptide. We also conducted coarse-grained (CG) molecular dynamics simulations to study the membrane curvature and the stabilization of ruptured membrane pores effect of TMD peptides. Our results indicated that TMD peptide decreases the local POPC lipids order. The membrane binding of TMD induced a positive membrane curvature, moreover, certain numbers of TMD could stabilize ruptured membrane pore in both CG and AA simulations. These results provide insight into the structure details of membrane pore formation by TMD peptides. The diameters of the pore are qualitatively in good agreement with available experimental data.

Specific Binding Protein ABCC1 Is Associated With Cry2Ab Toxicity in Helicoverpa armigera.

A pyramid strategy combining the crystal (Cry) 1A and 2A toxins in Bacillus thuringiensis (Bt) crops are active against many species of insects and nematode larvae. It has been widely used to delay pest adaption to genetically modified plants and broaden the insecticidal spectrum in many countries. Unfortunately, Cry2A can also bind with the specific receptor proteins of Cry1A. ATP-binding cassette (ABC) transporters can interact with Cry1A toxins as receptors in the insect midgut, and ABC transporter mutations result in resistance to Bt proteins. However, there is limited knowledge of the ABC transporters that specifically bind to Cry2Ab. Here, we cloned the ABCC1 gene in Helicoverpa armigera, which expressed at all larval stages and in nine different tissues. Expression levels were particularly high in fifth-instar larvae and Malpighian tubules. The two heterologously expressed HaABCC1 transmembrane domain peptides could specifically bind to Cry2Ab with high affinity levels. Moreover, transfecting HaABCC1 into the Spodoptera frugiperda nine insect cell significantly increased its mortality when exposed to Cry2Ab in vitro, and silencing HaABCC1 in H. armigera by RNA interference significantly reduced the mortality of larvae exposed to Cry2Ab in vivo. Altogether current results suggest that HaABCC1 serves as a functional receptor for Cry2Ab.

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13C-13C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may facilitate the transition of the membrane from hemifusion intermediates to the fusion pore.

The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

We propose mechanisms by which the transmembrane domain of vesicular stomatitis virus (VSV-TMD) promotes both initiation of fusion and formation of a fusion pore. Time courses of polyethyleneglycol (PEG)-mediated fusion of 25 nm small unilamellar vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), bovine brain sphingomyelin, and cholesterol (35:30:15:20 molar ratio) were recorded at pH 7.4 at five different temperatures (from 17°C to 37°C) and compared with time courses obtained with the same vesicles containing the fusion-active TMD of the G protein of VSV. Multiple time courses were fitted globally to a one-intermediate ensemble kinetic model to estimate the rate constants for conversion of the aggregated state to an intermediate hemifused state (k1, stalk, or I1) that rapidly transits to an unstable intermediate (I2 state) that converts to a final fusion pore state with a combined rate k3. The probabilities of lipid mixing, contents mixing, and contents leakage in the three states were also obtained from this analysis. The activation thermodynamics for each step were consistent with previously published models of lipid rearrangements during intermediate and pore formation. The influences of VSV-TMD, hexadecane, and VSV-TMD + hexadecane on the kinetics, activation thermodynamics, and membrane structure support the hypothesis that these two agents do not catalyze fusion by a common mechanism, except possibly at the lowest temperatures examined. VSV-TMD primarily catalyzed initial intermediate formation, although it substantially increased the probability of contents mixing in the intermediate state. Our results support the hypothesis that the catalytic influence of VSV-TMD on the initial-intermediate- and pore-forming steps of PEG-mediated fusion derives from its ability to impose a positive intrinsic curvature and thereby stress small unilamellar vesicle outer leaflets as well as the periphery of intermediate microstructures.

Peptides derived from the transmembrane domain of Bcl-2 proteins as potential mitochondrial priming tools.

The Bcl-2 family of proteins is crucial for apoptosis regulation. Members of this family insert through a specific C-terminal anchoring transmembrane domain (TMD) in the mitochondrial outer membrane where they hierarchically interact to determine cell fate. While the mitochondrial membrane has been proposed to actively participate in these protein-protein interactions, the influence of the TMD in the membrane-mediated interaction is poorly understood. Synthetic peptides (TMD-pepts) corresponding to the putative TMD of antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, and Mcl-1) and pro-apoptotic (Bax, Bak) members were synthesized and characterized. TMD-pepts bound more efficiently to mitochondria-like bilayers than to plasma membrane-like bilayers, and higher binding correlated with greater membrane perturbation. The Bcl-2 TMD peptides promoted mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release from isolated mitochondria and different cell lines. TMD-pepts exhibited nonapoptotic pro-death activity when apoptosis stimuli were absent. In addition, the peptides enhanced the apoptotic pathway induced by chemotherapeutic agents in cotreatment. Overall, the membrane perturbation effects of the TMD-pepts observed in the present study open the way for their use as new chemical tools to sensitize tumor cells to chemotherapeutic agents, in accordance with the concept of mitochondria priming.

A conserved cationic motif enhances membrane binding and insertion of the chloride intracellular channel protein 1 transmembrane domain.

The chloride intracellular channel protein 1 (CLIC1) is unique among eukaryotic ion channels in that it can exist as either a soluble monomer or an integral membrane channel. CLIC1 contains no known membrane-targeting signal sequences and the environmental factors which promote membrane binding of the transmembrane domain (TMD) are poorly understood. Here we report a positively charged motif at the C-terminus of the TMD and show that it enhances membrane partitioning and insertion. A 30-mer TMD peptide was synthesized in which the positively charged motif was replaced by three glutamate residues. The peptide was examined in 2,2,2-trifluoroethanol (TFE), sodium dodecyl sulfate micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using size-exclusion chromatography, far-UV CD, and fluorescence spectroscopy. The motif appears to enhance membrane interaction via electrostatic contacts and functions as an electrostatic plug to anchor the TMD in membranes. In addition, the motif is also involved in orientating the TMD with respect to the cis and trans faces of the membrane. These findings shed light on the intrinsic and environmental factors that promote the spontaneous conversion of CLIC1 from a water-soluble to a membrane-bound protein.

Functional Significance of K+ Channel β-Subunit KCNE3 in Auditory Neurons

The KCNE3 β-subunit interacts with and regulates the voltage-dependent gating, kinetics, and pharmacology of a variety of Kv channels in neurons. Because a single neuron may express multiple KCNE3 partners, it is impossible to predict the overall functional relevance of the single transmembrane domain peptide on the pore-forming K(+) channel subunits with which it associates. In the inner ear, the role of KCNE3 is undefined, despite its association with Meniere disease and tinnitus. To gain insights on the functional significance of KCNE3 in auditory neurons, we examined the properties of spiral ganglion neurons (SGNs) in Kcne3 null mutant neurons relative to their age-matched controls. We demonstrate that null deletion of Kcne3 abolishes characteristic wide variations in the resting membrane potentials of SGNs and yields age-dependent alterations in action potential and firing properties of neurons along the contour of the cochlear axis, in comparison with age-matched wild-type neurons. The properties of basal SGNs were markedly altered in Kcne3(-/-) mice compared with the wild-type controls; these include reduced action potential latency, amplitude, and increased firing frequency. Analyses of the underlying conductance demonstrate that null mutation of Kcne3 results in enhanced outward K(+) currents, which is sufficient to explain the ensuing membrane potential changes. Additionally, we have demonstrated that KCNE3 may regulate the activity of Kv4.2 channels in SGNs. Finally, there were developmentally mediated compensatory changes that occurred such that, by 8 weeks after birth, the electrical properties of the null mutant neurons were virtually indistinguishable from the wild-type neurons, suggesting that ion channel remodeling in auditory neurons progresses beyond hearing onset.

Side-Chain to Main-Chain Hydrogen Bonding Controls the Intrinsic Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix

Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer’s disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.

The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

The Transmembrane Domain Peptide of Vesicular Stomatitis Virus Promotes Both Intermediate and Pore Formation during PEG-Mediated Vesicle Fusion

A Lys–Trp Cation−π Interaction Mediates the Dimerization and Function of the Chloride Intracellular Channel Protein 1 Transmembrane Domain

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The oligomerization of the transmembrane domain (TMD) remains speculative despite it being implicated in pore formation. The extent to which electrostatic and van der Waals interactions drive folding and association of the dimorphic TMD is unknown and is complicated by the requirement of interactions favorable in both aqueous and membrane environments. Here we report a putative Lys37-Trp35 cation-π interaction and show that it stabilizes the dimeric form of the CLIC1 TMD in membranes. A synthetic 30-mer peptide comprising a K37M TMD mutant was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using far-ultraviolet (UV) circular dichroism, fluorescence, and UV absorbance spectroscopy. Our data suggest that Lys37 is not implicated in the folding, stability, or membrane insertion of the TMD peptide. However, removal of this residue impairs the formation of dimers and higher-order oligomers. This is accompanied by a 30-fold loss of chloride influx activity, suggesting that dimerization modulates the rate of chloride conductance. We propose that, within membranes, individual TMD helices associate via a Lys37-mediated cation-π interaction to form active dimers. The latter findings are also supported by results of modeling a putative TMD dimer conformation in which Lys37 and Trp35 form cation-π pairs at the dimer interface. Dimeric helix bundles may then associate to form fully active ion channels. Thus, within a membrane-like environment, aromatic interactions involving a polar lysine side chain provide a thermodynamic driving force for helix-helix association.

The backbone dynamics of the amyloid precursor protein transmembrane helix provides a rationale for the sequential cleavage mechanism of γ-secretase.

The etiology of Alzheimer's disease depends on the relative abundance of different amyloid-β (Aβ) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease γ-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a diglycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate: First, the GlyGly hinge may precisely position the substrate within γ-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region.

Investigating the Interaction Between Hcf106 Peptides and the Phospholipid Bilayer by Solid-State NMR Spectroscopy

In plant cells most thylakoid lumen proteins are synthesized in the cytosol as higher molecular weight precursors containing targeting sequences, which are imported into chloroplasts and localized to thylakoid lumen for function. Two thylakoid transport systems direct precursors to the thylakoid lumen. Our lab focuses on one of those transport systems: cpTat (chloroplast Twin arginine translocation) system, which transports fully folded proteins utilizing the trans-thylakoidal proton motive force as its only energy source. The cpTat pathway consists of three membrane-bound subunits, Tha4, Hcf106, and cpTatC. cpTatC and Hcf106 form a receptor complex where the precursor binds to initiate the translocation events, whereas Tha4 is initially found as a separate complex that joins the receptor once the precursor has bound. Hcf106 is predicted to contain a N-terminal transmembrane domain (TMD), followed by an amphipathic helix (APH) and an unstructured C-terminus. However, detailed structural information of Hcf106 remains unknown. In this study, peptides encompassing the Hcf106 TMD or APH domain were incorporated into POPC and POPC/MGDG vesicles and analyzed by solid-state NMR spectroscopy. Using 31P NMR we show that Hcf106 TMD/APH interact with the phospholipid headgroups. 2H-NMR revealed the perturbation of the deuterated acyl chain by the peptides. Lastly we used CD spectroscopy to analyze the secondary structure of Hcf106 TMD and APH peptides. The experimental results suggest: (1) Hcf106 TMD and APH peptides interact with both POPC and POPC/MGDG lipids with the lipid composition influencing the peptide-lipid interaction. (2) Hcf106 TMD and APH form alpha helical structures to varying degrees under the same conditions. (3) Hcf106 APH appears to be parallel to the membrane surface with a slight tilt into the hydrophobic region of phospholipids, whereas the TMD peptide appears to be perpendicular to the membrane surface.

Multiple γ-secretase product peptides are coordinately increased in concentration in the cerebrospinal fluid of a subpopulation of sporadic Alzheimer’s disease subjects

BackgroundAlcadeinα (Alcα) is a neuronal membrane protein that colocalizes with the Alzheimer's amyloid-β precursor protein (APP). Successive cleavage of APP by β- and γ-secretases generates the aggregatable amyloid-β peptide (Aβ), while cleavage of APP or Alcα by α- and γ-secretases generates non-aggregatable p3 or p3-Alcα peptides. Aβ and p3-Alcα can be recovered from human cerebrospinal fluid (CSF). We have previously reported alternative processing of APP and Alcα in the CSF of some patients with sporadic mild cognitive impairment (MCI) and AD (SAD).ResultsUsing the sandwich enzyme-linked immunosorbent assay (ELISA) system that detects total p3-Alcα, we determined levels of total p3-Alcα in CSF from subjects in one of four diagnostic categories (elderly controls, MCI, SAD, or other neurological disease) derived from three independent cohorts. Levels of Aβ40 correlated with levels of total p3-Alcα in all cohorts.ConclusionsWe confirm that Aβ40 is the most abundant Aβ species, and we propose a model in which CSF p3-Alcα can serve as a either (1) a nonaggregatable surrogate marker for γ-secretase activity; (2) as a marker for clearance of transmembrane domain peptides derived from integral protein catabolism; or (3) both. We propose the specification of an MCI/SAD endophenotype characterized by co-elevation of levels of both CSF p3-Alcα and Aβ40, and we propose that subjects in this category might be especially responsive to therapeutics aimed at modulation of γ-secretase function and/or transmembrane domain peptide clearance. These peptides may also be used to monitor the efficacy of therapeutics that target these steps in Aβ metabolism

HIV-1 gp41 Transmembrane Domain Interacts with the Fusion Peptide: Implication in Lipid Mixing and Inhibition of Virus–Cell Fusion

Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.

HIV gp41 Trans-Membrane Domain Promotes both Stalk and Fusion Pore Formation in Poly(Ethylene-) Glycol Mediated Membrane Fusion

The trans-membrane domain (TMD) of gp41 is essential for efficient fusion between HIV-1 and its host cell in vivo. HIV virus with gp41 mutated by R696L is reported to be defective in infectivity and fusion (Helseth, J Vir, 1990, 6314), so we have examined both native and R696L gp41 TMDs’ effects on PEG-mediated fusion of PC/PE/SM/CH (35/30/15/20) SUVs. Lipid mixing (LM), contents mixing (CM) and leakage (L) time courses were fitted globally to a 3-state sequential model (Weinreb & Lentz, BJ, 2007, 4012), from which we obtained estimates of rate constants for conversion between states as well as probabilities of LM, CM and L for each state. The WT peptide increased the rates of stalk (k1) and fusion pore (k3) formation in a cooperative fashion (maximum effect seen at ∼ 0.25mol% peptide or 6 peptides/vesicle). R696L peptide had no effect on k3 and a very minor effect on k1. At this concentration, CD spectroscopy showed the WT peptide to be ∼ 39% helix, 39% unordered, and 22% β-sheet, but to increase in β- and decrease in α-content at high peptide/lipid ratios up to 1/50. Neither peptide affected the extent of content mixing, but the R696L mutant actually inhibited the extent of lipid mixing. The native but not the mutant peptide increased the probability of content mixing in the stalk intermediate. Studies with hexadecane as a space-filling agent showed that the TMD peptide was unlikely to promote fusion by this mechanism. Analysis of transition state thermodynamics suggests that the TMD may disrupt interfacial packing so as to promote penetration of lipid acyl chains into the inter-bilayer space as a means of catalyzing stalk formation. Supported by NIGMS grant 32707 to BRL.

Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) are believed to be important players in RTK signal transduction. However, the degree of specificity and promiscuity of RTK TM domain lateral interactions in mammalian membranes has not been assessed in detail in the literature. A technique to probe the occurrence of interactions between TM domains and their biological significance is to evaluate the propensity for formation of heterodimers of a full-length RTK and its TM domain. Here we examine if the inhibition of two RTK pathogenic mutants, Neu/V664E and FGFR3/A391E, can be achieved by the TM domains of Neu, Neu/V664E, FGFR3 and FGFR3/A391E. We show that the TM domain of Neu/V664E specifically inhibits the phosphorylation of full-length Neu/V664E, while the wild-type Neu TM domain does not. In addition, Neu/V664E TM domain does not affect the phosphorylation levels of full-length FGFR3/A391E. The results suggest that TM domain peptides could be exploited in the future for the development of specific inhibitors of mutant RTKs.

Lipid Phase Specific Organisation of the Transmembrane Anchor of Influenza Hemagglutinin

Correct transport and localization of membrane proteins to distinct cellular organelles are crucial for cell growth but also for the infection cycle of enveloped viruses. Viral membrane proteins like hemagglutinin of influenza virus, anchored in the membrane via a single transmembrane domain (TMD), are transported to the plasma membrane and - together with other virus components - recruited to the budding site. Cholesterol and glycosphingolipid-enriched membrane microdomains are considered as assembly and budding sites for enveloped viruses such as influenza virus. Besides a most likely location of a sorting signal within the TMD sequence also the width of the bilayer is important, which is, amongst other factors, controlled by the cholesterol content of the membrane.Using different fluorescent-based approaches we study the impact of membrane hydrophobic thickness and membrane packing properties on the sorting behavior of virus derived TMD peptides containing a Trp residue in its center. The emission λmax depending on the hydrophobicity of the surrounding of the Trp residue and parallax analysis of fluorescence quenching are used to determine the Trp location in the lipid bilayer revealing a transmembrane orientation. Membrane thickness is altered by the use of lipids having different acyl chain length and also by the addition of cholesterol or decane shifting λmax. Incorporation of Rhodamine labeled TMD into giant unilamellar vesicles prepared from lipids with varying length and also from ternary lipid mixtures forming distinct liquid phases allows us to study phase dependent TMD localization by fluorescence microscopy. Typically, in the model system the TMD sorts into the liquid disordered phase in contrary to the raft association of hemagglutinin in cells. To address the latter by a more appropriate system, we used viral lipids for vesicle preparation mimicking the natural environment of the TMD.