RESUMEN

Nanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins. We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality.

Asunto(s)

Proteínas de la Membrana , Nanopartículas , Sistema Libre de Células/metabolismo , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Nanopartículas/química , Saposinas/química

RESUMEN

The initial step in the preparation of nanodiscs is to express and purify the membrane scaffold protein (MSP) to homogeneity. Current methods used for the isolation and purification of MSP utilize nickel affinity chromatography. However, the presence of a polyhistidine tag on the MSP often interferes with downstream steps where nanodiscs reconstituted with protein need to be isolated from empty ones. Therefore, one must engage in the finicky process of removing the polyhistidine tag from the MSP using a protease before the formation of nanodiscs. Herein, we describe a robust streamlined approach to produce tagless MSP by expression as inclusion bodies followed by cleavage with cyanogen bromide, and purification by gel filtration chromatography. In addition, the MSP prepared is devoid of tryptophan residues which facilitates tryptophan-based spectroscopic studies of reconstituted proteins. Dynamic light scattering and transmission electron microscopy showed that the tagless MSP produced was competent to produce nanodiscs.

Asunto(s)

Histidina/química , Proteínas de la Membrana/aislamiento & purificación , Nanoestructuras/química , Cromatografía de Afinidad , Proteínas de la Membrana/química , Níquel/química

RESUMEN

Lipid-bilayer nanodiscs (NDs) wrapped in membrane scaffold proteins (MSPs) have primarily been used to study membrane proteins of interest in a physiological environment. Recently, NDs have been employed in broader applications including drug delivery, cancer immunotherapy, bio-imaging, and therapeutic virucides. Here, we developed a method to synthesize a dimeric nanodisc, whose MSPs are circularly end-spliced, with long-term thermal stability and resistance to aggregation. The end-spliced nanodiscs (esNDs) were assembled using MSPs that were self-circularized inside the cytoplasm ofEscherichia colivia highly efficient protein trans-splicing. The esNDs demonstrated a consistent size and 4-5-fold higher stability against heat and aggregation than conventional NDs. Moreover, cysteine residues on trans-spliced circularized MSPs allowed us to modulate the formation of either monomeric nanodiscs (essNDs) or dimeric nanodiscs (esdNDs) by controlling the oxidation/reduction conditions and lipid-to-protein ratios. When the esdNDs were used to prepare an antiviral nanoperforator that induced the disruption of the viral membrane upon contact, antiviral activity was dramatically increased, suggesting that the dimerization of nanodiscs led to cooperativity between linked nanodiscs. We expect that controllable structures, long-term stability, and aggregation resistance of esNDs will aid the development of novel versatile membrane-mimetic nanomaterials with flexible designs and improved therapeutic efficacy.

Asunto(s)

Antivirales/uso terapéutico , Proteínas de la Membrana/uso terapéutico , Nanoestructuras/uso terapéutico , Animales , Antivirales/química , Escherichia coli/genética , Femenino , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/uso terapéutico , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Nanoestructuras/química , Orthomyxoviridae/efectos de los fármacos , Trans-Empalme , Envoltura Viral/efectos de los fármacos

RESUMEN

We describe the construction, expression and purification of three new membrane scaffold proteins (MSP) for use in assembling Nanodiscs. These new MSPs have a variety of luminescent properties for use in combination with several analytical methods. "Dark" MSP has no tryptophan residues, "Ultra-Dark" replaces both tryptophan and tyrosine with non-fluorescent side chains, and "Ultra-Bright" adds additional tryptophans to the parent membrane scaffold protein to provide a dramatic increase in native tryptophan fluorescence. All MSPs were used to successfully assemble Nanodiscs nominally 10 nm in diameter, and the resultant bilayer structure was characterized. An example of the usefulness of these new scaffold proteins is provided.

Asunto(s)

Péptidos Catiónicos Antimicrobianos/química , Colorantes Fluorescentes/química , Proteínas de la Membrana/química , Triptófano/química , Tirosina/química , Secuencia de Aminoácidos , Membrana Dobles de Lípidos/química , Unión Proteica , Multimerización de Proteína , Espectrometría de Fluorescencia

RESUMEN

Nanodiscs are discoidal protein-lipid complexes that have wide applications in membrane protein studies. Modeling and simulation of nanodiscs are challenging due to the absence of structures of many membrane scaffold proteins (MSPs) that wrap around the membrane bilayer. We have developed CHARMM-GUI Nanodisc Builder (http://www.charmm-gui.org/input/nanodisc) to facilitate the setup of nanodisc simulation systems by modeling the MSPs with defined size and known structural features. A total of 11 different nanodiscs with a diameter from 80 to 180 Å are made available in both the all-atom CHARMM and two coarse-grained (PACE and Martini) force fields. The usage of the Nanodisc Builder is demonstrated with various simulation systems. The structures and dynamics of proteins and lipids in these systems were analyzed, showing similar behaviors to those from previous all-atom and coarse-grained nanodisc simulations. We expect the Nanodisc Builder to be a convenient and reliable tool for modeling and simulation of nanodisc systems. © 2019 Wiley Periodicals, Inc.

Asunto(s)

Simulación por Computador , Lípidos/química , Modelos Químicos , Nanopartículas/química , Proteínas/química

RESUMEN

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aß40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aß40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 µM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aß40 aggregation and were also found to be suitable to trap Aß40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aß40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central ß-sheet-forming residues of Aß40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aß40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aß40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aß40 intermediates for high-resolution structural and neurobiological studies.

Asunto(s)

Péptidos beta-Amiloides/química , Materiales Biomiméticos/farmacología , Péptidos/farmacología , Agregado de Proteínas/efectos de los fármacos , Péptidos beta-Amiloides/antagonistas & inhibidores , Materiales Biomiméticos/química , Dicroismo Circular , Humanos , Cinética , Simulación de Dinámica Molecular , Nanoestructuras , Péptidos/química , Conformación Proteica , Conformación Proteica en Lámina beta/efectos de los fármacos

RESUMEN

Membrane scaffold proteins (MSPs) are synthetic derivatives of apolipoprotein A-I, a major protein component of human high-density lipoprotein complexes. The most common among these is the variant MSP1D1, which has been in the focus of research on membrane mimetics in the past. As such, the amphipathic MSP1D1 has the ability to self-assemble in the presence of synthetic phospholipids into discoidal nanoparticles, so called nanodiscs. The recombinant production of MSP is exclusively reported using a standard laboratory expression system of the pET family. However, strong variations in both yield and achieved concentration as well as complications related to unspecific degradation are commonly reported. In addition, the time-course of recombinant protein as well as specific protein yields have not yet been quantified conclusively. In this study, the time-course of MSP1D1 concentration was investigated in a standard pET expression system in terms of quantification of production and degradation rates in comparison to a reference protein (eGFP).

RESUMEN

Nanodiscs are membrane mimetics which may be used as tools for biochemical and biophysical studies of a variety of membrane proteins. These nanoscale structures are composed of a phospholipid bilayer held together by an amphipathic membrane scaffold protein (MSP). In the past, nanodiscs were successfully assembled with membrane scaffold protein 1D1 and 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine with a homogeneous diameter of ∼10 nm. In this study, the formation of nanoscale particles from MSP1D1 and rhamnolipid biosurfactants is investigated. Different protein to lipid ratios of 1:80, 1:90 and 1:100 were used for the assembly reaction, which were consecutively separated, purified and analyzed by size-exclusion chromatography (SEC) and dynamic light scattering (DLS). Size distributions were measured to determine homogeneity and confirm size dimensions. In this study, first evidence is presented on the formation of nanoscale particles with rhamnolipid biosurfactants and membrane scaffold proteins.

RESUMEN

The "resolution revolution" in electron cryomicroscopy (cryo-EM) profoundly changed structural biology of membrane proteins. Near-atomic structures of medium size to large membrane protein complexes can now be determined without crystallization. This significantly accelerates structure determination and also the visualization of small bound ligands. There is an additional advantage: the structure of membrane proteins can now be studied in their native or nearly native lipid bilayer environment. A popular lipid bilayer mimetic are lipid nanodiscs, which have been thoroughly characterized and successfully utilized in multiple applications. Here, we provide a guide for using lipid nanodiscs as a tool for single-particle cryo-EM of membrane proteins. We discuss general methodological aspects and specific challenges of protein reconstitution into lipid nanodiscs and high-resolution structure determination of the nanodisc-embedded complexes. Furthermore, we describe in detail case studies of two successful applications of nanodiscs in cryo-EM, namely, the structure determination of the rabbit ryanodine receptor, RyR1, and the pore-forming TcdA1 toxin subunit from Photorhabdus luminescens. We discuss cryo-EM-specific hurdles concerning sample homogeneity, distribution of reconstituted particles in vitreous ice, and solutions to overcome them.

Asunto(s)

Microscopía por Crioelectrón/métodos , Proteínas de la Membrana/química , Nanoestructuras/química , Animales , Toxinas Bacterianas/química , Microscopía por Crioelectrón/instrumentación , Membrana Dobles de Lípidos/química , Lípidos/química , Modelos Moleculares , Estructura Molecular , Conejos , Canal Liberador de Calcio Receptor de Rianodina/química

RESUMEN

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.

Asunto(s)

Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/aislamiento & purificación , Arabidopsis/genética , Biosíntesis de Proteínas , Proteínas RGS/biosíntesis , Proteínas RGS/aislamiento & purificación , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Proteínas RGS/química , Proteínas RGS/genética

RESUMEN

The changes in the orientation and conformation of three different membrane scaffold proteins (MSPs) upon entrapment in sol-gel-derived mesoporous silica monoliths were investigated. MSPs were examined in either a lipid-free or a lipid-bound conformation, where the proteins were associated with lipids to form nanolipoprotein particles (NLPs). NLPs are water-soluble, disk-shaped patches of a lipid bilayer that have amphiphilic MSPs shielding the hydrophobic lipid tails. The NLPs in this work had an average thickness of 5 nm and diameters of 9.2, 9.7, and 14.8 nm. We have previously demonstrated that NLPs are more suitable lipid-based structures for silica gel entrapment than liposomes because of their size compatibility with the mesoporous network (2-50 nm) and minimally altered structure after encapsulation. Here we further elaborate on that work by using a variety of spectroscopic techniques to elucidate whether or not different MSPs maintain their protein-lipid interactions after encapsulation. Fluorescence spectroscopy and quenching of the tryptophan residues with acrylamide, 5-DOXYL-stearic acid, and 16-DOXYL-stearic acid were used to determine the MSP orientation. We also utilized fluorescence anisotropy of tryptophans to measure the relative size of the NLPs and MSP aggregates after entrapment. Finally, circular dichroism spectroscopy was used to examine the secondary structure of the MSPs. Our results showed that, after entrapment, all of the lipid-bound MSPs maintained orientations that were minimally changed and indicative of association with lipids in NLPs. The tryptophan residues appeared to remain buried within the hydrophobic core of the lipid tails in the NLPs and appropriately spaced from the bilayer center. Also, after entrapment, lipid-bound MSPs maintained a high degree of α-helical content, a secondary structure associated with protein-lipid interactions. These findings demonstrate that NLPs are capable of serving as viable hosts for functional integral membrane proteins in the synthesis of sol-gel-derived bioinorganic hybrid nanomaterials.

Asunto(s)

Proteínas de la Membrana/química , Gel de Sílice/química , Acrilamida/química , Secuencia de Aminoácidos , Anisotropía , Dicroismo Circular , Lipoproteínas/química , Datos de Secuencia Molecular , Nanopartículas/química , Porosidad , Soluciones , Espectrometría de Fluorescencia , Triptófano/química

RESUMEN

Surface plasmon resonance (SPR) spectroscopy is a rapidly developing technique for the study of ligand binding interactions with membrane proteins, which are the major molecular targets for validated drugs and for current and foreseeable drug discovery. SPR is label-free and capable of measuring real-time quantitative binding affinities and kinetics for membrane proteins interacting with ligand molecules using relatively small quantities of materials and has potential to be medium-throughput. The conventional SPR technique requires one binding component to be immobilised on a sensor chip whilst the other binding component in solution is flowed over the sensor surface; a binding interaction is detected using an optical method that measures small changes in refractive index at the sensor surface. This review first describes the basic SPR experiment and the challenges that have to be considered for performing SPR experiments that measure membrane protein-ligand binding interactions, most importantly having the membrane protein in a lipid or detergent environment that retains its native structure and activity. It then describes a wide-range of membrane protein systems for which ligand binding interactions have been characterised using SPR, including the major drug targets G protein-coupled receptors, and how challenges have been overcome for achieving this. Finally it describes some recent advances in SPR-based technology and future potential of the technique to screen ligand binding in the discovery of drugs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.

Asunto(s)

Descubrimiento de Drogas , Proteínas de la Membrana/metabolismo , Resonancia por Plasmón de Superficie/métodos , Ligandos , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo

RESUMEN

Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/química , Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas Bacterianas/química , Proteínas Portadoras/química , Histidina/química , Fragmentos de Péptidos/química , Subunidades de Proteína/química , Salmonella typhimurium/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Transporte Biológico Activo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cationes Bivalentes , Escherichia coli/genética , Escherichia coli/metabolismo , Histidina/metabolismo , Hidrólisis , Magnesio/química , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimología , Homología de Secuencia de Aminoácido

RESUMEN

TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~200nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~3.5nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study ß-barrel membrane proteins in a membrane-like environment.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/química , Colicinas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Ferricromo/análogos & derivados , Membrana Dobles de Lípidos/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ferricromo/química , Expresión Génica , Cinética , Imitación Molecular , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Termodinámica

RESUMEN

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.

Asunto(s)

Endotelina-1/química , Liposomas/química , Receptor de Endotelina A/química , Receptor de Endotelina B/química , Sistema Libre de Células/metabolismo , Detergentes/química , Endotelina-1/metabolismo , Expresión Génica , Humanos , Cinética , Nanoestructuras/química , Unión Proteica , Pliegue de Proteína , Receptor de Endotelina A/biosíntesis , Receptor de Endotelina A/genética , Receptor de Endotelina B/biosíntesis , Receptor de Endotelina B/genética