RESUMO
By conducting a systematic study of model lipid membranes using the atomic force microscopy (AFM) indentation, we demonstrate the importance of an experimental protocol on the determination of their mechanical parameters. We refine the experimental approach by analyzing the influence of the contact mechanics models used to process the data, substrate preparation, and indenter geometry. We show that both bending rigidity and area compressibility can be determined from a single AFM indentation measurement.
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We present a reliable method for the fabrication of fluid phase, unsaturated lipid bilayers by self-assembly onto charged Self-Assembled Monolayer (SAM) surfaces with tunable membrane to surface aqueous interlayers. Initially, the formation of water interlayers between membranes and charged surfaces was characterized using a comparative series of bilayers deposited onto charged, self-assembled monolayers by sequential layer deposition. Using neutron reflectometry, a bilayer to surface water interlayer of â¼8 Å was found between the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane and an anionic carboxyl terminated grafted SAM with the formation of this layer attributed to bilayer repulsion by hydration water on the SAM surface. Furthermore, we found we could significantly reduce the technical complexity of sample fabrication through self-assembly of planar membranes onto the SAM coated surfaces. Vesicle fusion onto carboxyl-terminated monolayers yielded high coverage (>95%) bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) which floated on a 7-11 Å solution interlayer between the membrane and the surface. The surface to membrane distance was then tuned via the addition of 200 mM NaCl to the bulk solution immersing a POPC floating membrane, which caused the water interlayer to swell reversibly to â¼33 Å. This study reveals that biomimetic membrane models can be readily self-assembled from solution onto functionalized surfaces without the use of polymer supports or tethers. Once assembled, surface to membrane distance can be tailored to the experimental requirements using physiological concentrations of electrolytes. These planar bilayers only very weakly interact with the substrate and are ideally suited for use as biomimetic models for accurate in vitro biochemical and biophysical studies, as well as for technological applications, such as biosensors.
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A combined computational and experimental study of small unilamellar vesicle (SUV) fusion on mixed self-assembled monolayers (SAMs) terminated with different deuterated tether moieties (-(CD2)7CD3 or -(CD2)15CD3) is reported. Tethered bilayer lipid membrane (tBLM) formation of synthetic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine was initially probed on SAMs with controlled tether (d-alkyl tail) surface densities and lateral molecular packing using quartz crystal microbalance with dissipation monitoring (QCM-D). Long time-scale coarse-grained molecular dynamics (MD) simulations were then employed to elucidate the mechanisms behind the interaction between the SUVs and the different phases formed by the -(CD2)7CD3 and -(CD2)15CD3 tethers. Furthermore, a series of real time kinetics was recorded under different osmotic conditions using QCM-D to determine the accumulated lipid mass and for probing the fusion process. It is shown that the key factors driving the SUV fusion and tBLM formation on this type of surfaces involve tether insertion into the SUVs along with vesicle deformation. It is also evident that surface densities of the tethers as small as a few mol% are sufficient to obtain stable tBLMs with a high reproducibility. The described "sparsely tethered" tBLM system can be advantageous in studying different biophysical phenomena, such as membrane protein insertion, effects of receptor clustering, and raft formation.
Assuntos
Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Bicamadas Lipídicas/química , Reprodutibilidade dos Testes , Polímeros , Proteínas de MembranaRESUMO
Alpha and beta interferons (IFN-alpha and IFN-beta) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-alpha2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-beta. Compared to wild-type (wt) IFN-alpha2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-beta, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-beta, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-beta and higher levels of gene induction or repression than those for wt IFN-alpha2. Thus, we show that the differential activities of IFN-beta are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-alpha subtypes suggests that IFN-alpha has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-beta.
Assuntos
Interferon-alfa/genética , Interferon-alfa/farmacologia , Interferon beta/metabolismo , Interferon beta/farmacologia , Proteínas de Membrana/metabolismo , Receptores de Interferon/metabolismo , Antivirais/farmacologia , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Interferon-alfa/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Complexos Multiproteicos , Mutação , Regiões Promotoras Genéticas , Receptor de Interferon alfa e beta , Receptores de Interferon/efeitos dos fármacos , Receptores de Interferon/genética , Ativação TranscricionalRESUMO
Nanofluidic systems offer a huge potential for discovery of new molecular transport and chemical phenomena that can be employed for future technologies. Herein, we report on the transport behavior of surface-reactive compounds in a nanometer-scale flow of phospholipids from a scanning probe. We have investigated microscopic deposit formation on polycrystalline gold by lithographic printing and writing of 1,2-dioleoyl-sn-glycero-3-phosphocholine and eicosanethiol mixtures, with the latter compound being a model case for self-assembled monolayers (SAMs). By analyzing the ink transport rates, we found that the transfer of thiols was fully controlled by the fluid lipid matrix allowing to achieve a certain jetting regime, i.e., transport rates previously not reported in dip-pen nanolithography (DPN) studies on surface-reactive, SAM-forming molecules. Such a transport behavior deviated significantly from the so-called molecular diffusion models, and it was most obvious at the high writing speeds, close to 100 µm s-1. Moreover, the combined data from imaging ellipsometry, scanning electron microscopy, atomic force microscopy (AFM), and spectroscopy revealed a rapid and efficient ink phase separation occurring in the AFM tip-gold contact zone. The force curve analysis indicated formation of a mixed ink meniscus behaving as a self-organizing liquid. Based on our data, it has to be considered as one of the co-acting mechanisms driving the surface reactions and self-assembly under such highly nonequilibrium, crowded environment conditions. The results of the present study significantly extend the capabilities of DPN using standard AFM instrumentation: in the writing regime, the patterning speed was already comparable to that achievable by using electron beam systems. We demonstrate that lipid flow-controlled chemical patterning process is directly applicable for rapid prototyping of solid-state devices having mesoscopic features as well as for biomolecular architectures.
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Ouro/química , Tinta , Modelos Químicos , Fosfolipídeos/química , Impressão , Redação , Compostos de Sulfidrila/químicaRESUMO
Type I interferons (IFNs) elicit antiviral, antiproliferative and immunmodulatory responses by binding to a shared cell surface receptor comprising the transmembrane proteins ifnar1 and ifnar2. Activation of differential response patterns by IFNs has been observed, suggesting that members of the family play different roles in innate immunity. The molecular basis for differential signaling has not been identified yet. Here, we have investigated the recognition of various IFNs including several human IFNalpha species, human IFNomega and human IFNbeta as well as ovine IFNtau2 by the receptor subunits in detail. Binding to the extracellular domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC) was monitored in real time by reflectance interference and total internal reflection fluorescence spectroscopy. For all IFNs investigated, competitive 1:1 interaction not only with ifnar2-EC but also with ifnar1-EC was shown. Furthermore, ternary complex formation was studied with ifnar1-EC and ifnar2-EC tethered onto solid-supported membranes. These analyses confirmed that the signaling complexes recruited by IFNs have very similar architectures. However, differences in rate and affinity constants over several orders of magnitude were observed for both the interactions with ifnar1-EC and ifnar2-EC. These data were correlated with the potencies of ISGF3 activation, antiviral and anti-proliferative activity on 2fTGH cells. The ISGF3 formation and antiviral activity correlated very well with the binding affinity towards ifnar2. In contrast, the affinity towards ifnar1 played a key role for antiproliferative activity. A striking correlation was observed for relative binding affinities towards ifnar1 and ifnar2 with the differential antiproliferative potency. This correlation was confirmed by systematically engineering IFNalpha2 mutants with very high differential antiproliferative potency.
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Interferon Tipo I/metabolismo , Bicamadas Lipídicas/metabolismo , Receptores de Interferon/metabolismo , Transdução de Sinais , Humanos , Interferon Tipo I/química , Interferon Tipo I/genética , Interferon alfa-2 , Interferon-alfa/química , Interferon-alfa/genética , Ligantes , Bicamadas Lipídicas/química , Modelos Biológicos , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Interferon/química , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
We describe herein a series of self-assembled monolayers (SAMs) on gold designed for adjustable tethering of model lipid membrane phases. The SAMs consist of deuterated aliphatic anchors, HS(CH2)15CONH(CH2CH2O)6CH2CONH-X, where X is either -(CD2)7CD3 or -(CD2)15CD3, dispersed in a stable matrix of protein-repellent molecules, HS(CH2)15CONHCH2CH2OH. The mixed SAMs with variable surface densities of the anchors are thoroughly characterized before and after adsorption of phospholipids by means of ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy (IRRAS). In all cases, the bottom portions of the mixed SAMs (i.e., the h-alkyl thiol segments of the molecules) are arranged in a highly ordered all-trans conformation stabilized by a network of lateral hydrogen bonds. The terminal portions of the anchors (the oligo(ethylene glycol) spacer and deuterated alkyl segments, respectively), however, possess less ordered conformations in the mixed composition regime. For the SAMs containing the longer anchors (-(CD2)15CD3), the contact angle and infrared data point toward partial phase segregation. These findings are in excellent agreement with molecular dynamics simulations by Schulze and Stein. Upon analysis in air, the IRRAS data also indicate strong interaction between the adsorbed phospholipid molecules and the d-alkyl tails of the mixed SAM constituents. In such assemblies are the alkyl tails of the phospholipids aligned perpendicularly with respect to the supporting substrate, regardless of the anchor length. We also probed the in situ formation of a tethered bilayer lipid membrane (tBLM) via fusion of small unilamellar vesicles (SUVs) on the characterized SAMs using a quartz crystal microbalance with dissipation monitoring. Our experiments show that SUVs fuse efficiently of the two mixed SAMs with and without a pre-adsorbed lipid layer. Owing to the defined molecular composition and phase behavior, our SAM platform is attractive for detailed studies of tBLM formation and cell mimetic applications.
Assuntos
Amidas/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Compostos de Sulfidrila/química , Adsorção , Deutério/química , Ouro/química , Estrutura Molecular , Polietilenoglicóis/química , Técnicas de Microbalança de Cristal de Quartzo , Espectrofotometria InfravermelhoRESUMO
The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.
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Membrana Celular/metabolismo , Receptores Tipo II de Interleucina-4/metabolismo , Citoesqueleto de Actina , Compartimento Celular , Dimerização , Células HeLa , Humanos , Ligantes , Receptores Tipo II de Interleucina-4/agonistas , Fator de Transcrição STAT6/metabolismoRESUMO
Ligand-induced cross-linking of the type I interferon (IFN) receptor subunits ifnar1 and ifnar2 induces a pleiotrophic cellular response. Several studies have suggested differential signal activation by flexible recruitment of the accessory receptor subunit ifnar1. We have characterized the roles of the four Ig-like sub-domains (SDs) of the extracellular domain of ifnar1 (ifnar1-EC) for ligand recognition and receptor assembling. Various sub-fragments of ifnar1-EC were expressed in insect cells and purified to homogeneity. Solid phase binding assays with the ligands IFN(alpha)2 and IFN(beta) revealed that all three N-terminal SDs were required and sufficient for ligand binding, and that IFN(alpha)2 and IFN(beta) compete for this binding site. Cellular binding assays with different fragments, however, highlighted the key role of the membrane-proximal SD for the formation of an in situ IFN-receptor complex. Even substitution with the corresponding SD from homologous cytokine receptors did not restore high-affinity ligand binding. Receptor assembling analysis on supported lipid bilayers in vitro revealed that the membrane-proximal SD controls appropriate orientation of the receptor on the membrane, which is required for efficient association of ifnar1 into the ternary complex.
Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Receptores de Interferon/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Separação Celular , Cromatografia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Citometria de Fluxo , Glicosilação , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Cinética , Ligantes , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptor de Interferon alfa e beta , Homologia de Sequência de Aminoácidos , Transdução de Sinais , TransfecçãoRESUMO
Type I interferons (IFNs) elicit antiviral, antiproliferative and immuno-modulatory responses through binding to a shared receptor consisting of the transmembrane proteins ifnar1 and ifnar2. Differential signaling by different interferons, in particular IFNalphas and IFNbeta, suggests different modes of receptor engagement. Using reflectometric interference spectroscopy (RIfS), we studied kinetics and affinities of the interactions between IFNs and the extracellular receptor domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC). For IFNalpha2, we determined a K(D) value of 3 nM and 5 microM for the interaction with ifnar2-EC and ifnar1-EC, respectively. As compared to IFNalpha2, IFNbeta formed complexes with ifnar2-EC as well as ifnar1-EC with substantially higher affinity. For neither IFNalpha2 nor IFNbeta was stabilization of the complex with ifnar1-EC in the presence of soluble ifnar2-EC observed. We investigated ligand-induced complex formation with ifnar1-EC and ifnar2-EC being tethered onto solid-supported, fluid lipid bilayers by RIfS and total internal reflection fluorescence spectroscopy. We observed very stable binding of IFNalpha2 at high receptor surface concentrations with an apparent k(d) value approximately 200 times lower than that for ifnar2-EC alone. The apparent k(d) value was strongly dependent on the surface concentration of the receptor components, suggesting kinetic stabilization. This was corroborated by the fast exchange of labeled IFNalpha2 bound to the receptor by unlabeled IFNalpha2. Taken together, our results indicate that IFN first binds to ifnar2 and subsequently recruits ifnar1 in a transient fashion. In particular, this second step is much more efficient for IFNbeta than for IFNalpha2, which could explain differential activities observed for these IFNs.
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Bicamadas Lipídicas/metabolismo , Receptores de Interferon/metabolismo , Animais , Humanos , Interferons/metabolismo , Ligantes , Proteínas de Membrana , Polietilenoglicóis , Receptor de Interferon alfa e beta , Fatores de TempoRESUMO
We describe herein a platform to study protein-protein interactions and to form functional protein complexes in nanoscopic surface domains. For this purpose, we employed multivalent chelator (MCh) templates, which were fabricated in a stepwise procedure combining dip-pen nanolithography (DPN) and molecular recognition-directed assembly. First, we demonstrated that an atomic force microscope (AFM) tip inked with an oligo(ethylene glycol) (OEG) disulfide compound bearing terminal biotin groups can be used to generate biotin patterns on gold achieving line widths below 100 nm, a generic platform for fabrication of functional nanostructures via the highly specific biotin-streptavidin recognition. Subsequently, we converted such biotin/streptavidin patterns into functional MCh patterns for reversible assembly of histidine-tagged (His-tagged) proteins via the attachment of a tris-nitriloacetic acid (trisNTA) biotin derivative. Fluorescence microscopy confirmed reversible immobilization of the receptor subunit ifnar2-His10 and its interaction with interferon-alpha2 labeled with fluorescent quantum dots in a 7 x 7 dot array consisting of trisNTA spots with a diameter of approximately 230 nm. Moreover, we carried out characterization of the specificity, stability, and reversibility as well as quantitative real-time analysis of protein-protein interactions at the fabricated nanopatterns by imaging surface plasmon resonance. Our work offers a route for construction and analysis of functional protein-based nanoarchitectures.
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Nanopartículas/química , Nanotecnologia/métodos , Proteínas/química , Biotina/química , Quelantes/farmacologia , Etilenoglicol/química , Cinética , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Químicos , Nanoestruturas/química , Mapeamento de Interação de Proteínas , Ressonância de Plasmônio de SuperfícieRESUMO
The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFN alpha 2 and IFN beta induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFN alpha 2 or IFN beta with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.
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Interferon-alfa/química , Interferon beta/química , Receptor de Interferon alfa e beta/química , Transferência Ressonante de Energia de Fluorescência , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Microscopia Eletrônica de Transmissão , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptor de Interferon alfa e beta/metabolismo , TemperaturaRESUMO
Ligand-induced cross-linking of cell surface receptors is a basic paradigm of signal activation by many transmembrane receptors. After ligand binding, the receptor complexes formed on the membrane are dynamically maintained by two-dimensional protein-protein interactions on the membrane. The biophysical principles governing the dynamics of such interactions have not been understood, mainly because the measurement of lateral interactions on membranes so far has not been experimentally addressed. Here, we describe a generic approach for measuring two-dimensional dissociation rate constants in vitro using a novel high-affinity chelator lipid for reconstituting a ternary cytokine-receptor complex on solid-supported membranes. While monitoring the interaction between the ligand and one of the receptor subunits on the membrane by fluorescence resonance energy transfer, the equilibrium on the surface was perturbed by rapidly tethering a large excess of the unlabeled receptor subunit. Displacement of labeled by unlabeled protein in the ternary complex was detected as a recovery of the donor quenching. Since the dissociation of the ligand-receptor complex in plane of the membrane was the rate-limiting step under these conditions, the two-dimensional rate constant of this process was determined. Strikingly, the two-dimensional dissociation was much slower than ligand dissociation into solution, suggesting that membrane tethering significantly affects the dissociation process. This result highlights the importance of studying ligand-receptor complexes tethered to membranes for understanding the principles governing signal activation by ligand-induced receptor assembling.
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Citocinas/química , Lipídeos de Membrana/química , Membranas Artificiais , Receptores de Citocinas/química , Quelantes/química , Quelantes/metabolismo , Citocinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Interferon-alfa/química , Cinética , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , Ácido Nitrilotriacético/química , Receptor de Interferon alfa e beta , Receptores de Citocinas/metabolismo , Receptores de Interferon/químicaRESUMO
This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2-3 months.
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Membranas Artificiais , Receptores de Superfície Celular/metabolismo , Interferometria/métodos , Cinética , Ligantes , Luz , Espectrometria de FluorescênciaRESUMO
Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.
Assuntos
Corantes Fluorescentes/química , Histidina/química , Interferon-alfa/química , Proteínas de Membrana/química , Oligopeptídeos/química , Receptores de Interferon/química , Quelantes/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Moleculares , Ácido Nitrilotriacético/química , Fotoquímica , Receptor de Interferon alfa e beta , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
Ligand-receptor interactions within the plane of the plasma membrane play a pivotal role for transmembrane signaling. The biophysical principles of protein-protein interactions on lipid bilayers, though, have hardly been experimentally addressed. We have dissected the interactions involved in ternary complex formation by ligand-induced cross-linking of the subunits of the type I interferon (IFN) receptors ifnar1 and ifnar2 in vitro. The extracellular domains ifnar1-ectodomain (EC) and ifnar2-EC were tethered in an oriented manner on solid-supported lipid bilayers. The interactions of IFNalpha2 and several mutants, which exhibit different association and dissociation rate constants toward ifnar1-EC and ifnar2-EC, were monitored by simultaneous label-free detection and surface-sensitive fluorescence spectroscopy. Surface dissociation rate constants were determined by measuring ligand exchange kinetics, and by measuring receptor exchange on the surface by fluorescence resonance energy transfer. Strikingly, approximately three-times lower dissociation rate constants were observed for both receptor subunits compared to the dissociation in solution. Based on these directly determined surface-dissociation rate constants, the surface-association rate constants were assessed by probing ligand dissociation at different relative surface concentrations of the receptor subunits. In contrast to the interaction in solution, the association rate constants depended on the orientation of the receptor components. Furthermore, the large differences in association kinetics observed in solution were not detectable on the surface. Based on these results, the key roles of orientation and lateral diffusion on the kinetics of protein interactions in plane of the membrane are discussed.
Assuntos
Citocinas/metabolismo , Mapeamento de Interação de Proteínas , Receptores de Citocinas/metabolismo , Simulação por Computador , Transferência Ressonante de Energia de Fluorescência , Cinética , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Proteínas Recombinantes/metabolismoRESUMO
We describe an experimental approach for studying ligand-receptor interactions in the plane of the membrane. The extracellular domains of the type I interferon receptor subunits ifnar1-EC and ifnar2-EC were tethered in an oriented fashion onto solid-supported, fluid lipid bilayers, thus mimicking membrane anchoring and lateral diffusion of the receptor. Ligand-induced receptor assembling was investigated by simultaneous total internal reflection fluorescence spectroscopy and reflectance interferometry (RIf). Based on a rigorous characterization of the interactions of fluorescence-labeled IFNalpha2 with each of the receptor subunits, the dynamics of the ternary complex formation on the fluid lipid bilayer was addressed in further detail making use of the features of the simultaneous detection. All these measurements supported the formation of a ternary complex in two steps, i.e., association of the ligand to ifnar2-EC and subsequent recruitment of ifnar1-EC on the surface of the membrane. Based on the ability to control and quantify the receptor surface concentrations, equilibrium, and rate constants of the interaction in the plane of the membrane were determined by monitoring ligand dissociation at different receptor surface concentrations. Using mutants of IFNalpha2 binding to ifnar2-EC with different association rate constants, the key role of the association rate constants for the assembling mechanism was demonstrated.