RESUMO
Membrane proteins, such as G-protein coupled receptors, control communication between cells and their environments and are indispensable for many cellular functions. Nevertheless, structural studies on membrane proteins lag behind those on water-soluble proteins, due to their low structural stability, making it difficult to obtain crystals for X-ray crystallography. Optimizing conditions to improve the stability of membrane proteins is essential for successful crystallization. However, the optimization usually requires large amounts of purified samples, and it is a time-consuming and trial-and-error process. Here, we report a rapid method for precrystallization screening of membrane proteins using Clear Native polyacrylamide gel electrophoresis (CN-PAGE) with the modified Coomassie Brilliant Blue G-250 (mCBB) stain that was reduced in sodium formate. A2A adenosine receptor (A2AAR) was selected as a target membrane protein, for which we previously obtained the crystal structure using an antibody, and was expressed as a red fluorescent protein fusion for in-gel fluorescence detection. The mCBB CN-PAGE method enabled the optimization of the solubilization, purification, and crystallization conditions of A2AAR using the solubilized membrane fraction expressing the protein without purification procedures. These data suggest the applicability of mCBB CN-PAGE technique to a wide variety of integral membrane proteins.
Assuntos
Eletroforese em Gel de Poliacrilamida Nativa/métodos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Cristalografia por Raios X/métodos , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
We describe a detailed protocol for heterologous expression of the human adenosine A2A G-protein coupled receptor (GPCR), using Pichia pastoris. Details are also provided for the reconstitution and functional purification steps. Yields of 2-6 mg/g membrane were obtained prior to functional purification (ligand column purification). Typically, functional purification reduced overall yields by a factor of 2-4, resulting in final functional production of 0.5-3 mg/L membrane. Yeast is an excellent protein expression system for NMR given its high tolerance for isotope-enriched solvents and its ability to grow in minimal media.
Assuntos
Marcação por Isótopo/métodos , Pichia/genética , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/isolamento & purificação , Flúor , Expressão Gênica , Humanos , Microrganismos Geneticamente Modificados , Receptor A2A de Adenosina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. The studies presented in this paper also underline the importance of the protein's purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.
Assuntos
Ésteres do Colesterol/química , Ácidos Cólicos/química , Éteres Fosfolipídicos/química , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Proteínas Recombinantes/química , Humanos , Estrutura Secundária de ProteínaRESUMO
G protein-coupled receptors (GPCRs) are of particular importance for drug discovery, being the targets of many existing drugs, and being linked to many diseases where new therapies are required. However, as integral membrane proteins, they are generally unstable when removed from their membrane environment, precluding them from the wide range of structural and biophysical techniques which can be applied to soluble proteins such as kinases. Through the use of protein engineering methods, mutations can be identified which both increase the thermostability of GPCRs when purified in detergent, as well as biasing the receptor toward a specific physiologically relevant conformational state. The resultant stabilized receptor (known as a StaR) can be purified in multiple-milligram quantities, whilst retaining correct folding, thus enabling the generation of reagents suitable for a broad range of structural and biophysical studies. Example protocols for the purification of StaR proteins for analysis, ligand screening with the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM), surface plasmon resonance (SPR), and crystallization for structural studies are presented.
Assuntos
Fenômenos Biofísicos , Fracionamento Químico/métodos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/isolamento & purificação , Cristalização , Eletroforese em Gel de Poliacrilamida , Proteínas Imobilizadas/química , Proteínas Imobilizadas/isolamento & purificação , Proteínas Imobilizadas/metabolismo , Ligantes , Maleimidas/química , Estabilidade Proteica , Receptor A2A de Adenosina/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Xantinas/metabolismoRESUMO
Crystallization of G protein-coupled receptors (GPCRs) is successful due to the development of generic protein engineering strategies, which has resulted in the structure determination of more than 25 GPCRs, including representatives from class A, B, C, and F. Most of the X-ray structures available correspond to an inactive conformation of the receptor bound to an antagonist. Only a few high-resolution structures of agonist-bound conformations of GPCRs have been determined over the last 6 years. Here, we describe the purification and crystallization protocols of a thermostabilized agonist-bound conformation of the human adenosine A2A receptor.
Assuntos
Agonistas do Receptor A2 de Adenosina/metabolismo , Fracionamento Químico/métodos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Temperatura , Membrana Celular/metabolismo , Cristalização , Humanos , Conformação Proteica , Estabilidade Proteica , Receptor A2A de Adenosina/metabolismo , VolatilizaçãoRESUMO
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (~5°C) compared with detergent [DDM (n-dodecyl-ß-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([(3)H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.
Assuntos
Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Linhagem Celular , Células HEK293 , Humanos , Maltose/análogos & derivados , Maltose/química , Pichia/química , Pichia/genética , Pichia/metabolismo , Estabilidade Proteica , Receptor A2A de Adenosina/biossíntese , Receptor A2A de Adenosina/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras of the human ß(2)-adrenergic and human A(2A) adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b(562)RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in a significant improvement in stability and structure of GPCR-fusion constructs.
Assuntos
Citocromos b/química , Muramidase/química , Receptor A2A de Adenosina/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia em Gel , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Citocromos b/biossíntese , Citocromos b/isolamento & purificação , Humanos , Dados de Sequência Molecular , Muramidase/biossíntese , Muramidase/isolamento & purificação , Estabilidade Proteica , Receptor A2A de Adenosina/biossíntese , Receptor A2A de Adenosina/isolamento & purificação , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The isolation of membrane proteins with the aim of producing highly pure, homogeneous, stable, and functional material remains challenging, and it is often necessary to develop protein-specific purification protocols by trial and error. One key tool that is required in the development of a suitable protocol is a functional assay. This unit describes a range of different protocols for isolation of the human adenosine A2a receptor (A(2a)R). These protocols show the importance of developing a robust method for comparing the quality of protein obtained by a combination of biophysical analyses including SDS-PAGE, analytical size-exclusion chromatography, and functional analysis. One of the keys to isolating and maintaining a functional receptor, found not only in the optimal protocol described here but in other published examples, is that there should be no more than two chromatographic steps.
Assuntos
Pichia/química , Engenharia de Proteínas/métodos , Receptor A2A de Adenosina/isolamento & purificação , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Pichia/genética , Estabilidade Proteica , Proteólise , Receptor A2A de Adenosina/análise , Receptor A2A de Adenosina/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeRESUMO
New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.
Assuntos
Caenorhabditis elegans/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Bovinos , Expressão Gênica , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/isolamento & purificação , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rodopsina/genética , Rodopsina/isolamento & purificação , Rodopsina/metabolismoRESUMO
G-protein-coupled receptors (GPCRs) represent a diverse protein family of receptors that transduce signals from the extracellular surrounding to intracellular signaling molecules evoking various cellular responses. It is now widely accepted that GPCRs are expressed and function as dimers or most probably as oligomers of more than two receptor protomers. The heteromer has different biochemical and pharmacological characteristics from the monomers, which increases the functional responses of GPCRs. GPCRs are involved in many diseases, and are also the target of around half of all modern medicinal drugs. In the case of Parkinson's disease, a degenerative process caused by gradual disappearance of dopaminergic nigrostriatal neurons, it is suspected that the targets for treatment should be dopamine-receptor-containing heteromers. Technologies based on the use of fluorescent- or luminescent-fused receptors and adaptations of resonance energy transfer (RET) techniques have been useful in investigating the functional inter-relationships between receptors in a heteromer. In this study functional recombinant adenosine A(2A)-Rluc, dopamine D(2)-GFP(2) and histamine H(3)-YFP receptor fusion proteins were successfully cloned and characterized, producing the essential basis for heteromerization studies between these receptors. This might provide a better insight into their pharmacological and functional inter-relationships in the brain and enable the design and evaluation of new therapeutic strategies for Parkinson's disease.
Assuntos
Multimerização Proteica/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes de Fusão/síntese química , Animais , Proteínas de Bactérias/genética , Células CHO , Cricetinae , Cricetulus , Agonistas de Dopamina/farmacologia , Desenho de Fármacos , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/isolamento & purificação , Receptores de Dopamina D2/química , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/isolamento & purificação , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos H3/química , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/isolamento & purificação , Proteínas Recombinantes de Fusão/genéticaRESUMO
Recent high resolution structures of modified G-protein coupled receptors (GPCRs) have provided major insight into the mechanisms of receptor-ligand binding. However understanding of the complete mechanism of GPCR function remains limited. This study characterised C-terminally truncated versions of the human adenosine A(2A) receptor (A(2A)R) with a view to producing protein suitable for structural studies. The constructs terminated at residue A316, removing the intracellular C-terminal tail, or V334, producing a C-terminal tail equivalent in length to that of rhodopsin. Higher levels of functional receptor before and after solubilisation were obtained for both C-terminally truncated constructs compared to the wild-type receptor (WT) as assessed by radioligand binding analysis using [(3)H]ZM241385. The construct which yielded the highest level of functional receptor, V334 A(2A)R, was purified in DDM to high homogeneity with a final yield of 2 mg/L. Binding analysis revealed that the purified receptor had a specific activity of 20.2+/-1.2 nmol/mg, close to the theoretical maximum. Pure V334 A(2A)R was resistant to degradation over 15 days when stored at 4 degrees C or 20 degrees C and showed remarkable functional stability when stored at 4 degrees C, retaining 84% of initial functionality after 30 days. This construct is an excellent candidate for structural studies.
Assuntos
Receptor A2A de Adenosina/isolamento & purificação , Receptor A2A de Adenosina/metabolismo , Linhagem Celular , Ésteres do Colesterol/metabolismo , Detergentes , Expressão Gênica , Humanos , Estabilidade Proteica , Receptor A2A de Adenosina/genética , SolubilidadeRESUMO
The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (approximately 4mg/L of culture) of functional human adenosine A(2)a receptor fused to a green fluorescent protein (A(2)aR-GFP) from Saccharomyces cerevisiae. In this work, we re-engineered A(2)aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A(2)aR and A(2)aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A(2)aR-GFP-His(10). One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-beta-d-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A(2)aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A(2)a-His(10) receptor was purified in quantities of 6+/-2mg/L of culture, with ligand-binding yields of 1mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A(2)aR yet achieved from any heterologous expression system.
Assuntos
Receptor A2A de Adenosina , Saccharomyces cerevisiae/metabolismo , Colesterol/metabolismo , Clonagem Molecular , Detergentes/química , Humanos , Micelas , Conformação Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/isolamento & purificação , Receptor A2A de Adenosina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
A glycosylation deficient (dG) version of the human adenosine 2a receptor (hA2aR) was made in Pichia pastoris strain SMD1163. Under optimal conditions, expression levels of between 8 and 12pmol receptor/mg membrane protein were obtained routinely. In a shake flask, this is equivalent to ca. 0.2mg of receptor per litre of culture. The level of functional receptor produced was essentially independent of the pH of the yeast media. In contrast to this, addition of the hA2aR antagonist theophylline to the culture media caused a twofold increase in receptor expression. A similar effect on dG hA2aR production was also observed when the induction temperature was reduced from 29 to 22 degrees C. In P. pastoris membranes, dG hA2aR had native-like pharmacological properties, binding antagonists with rank potency ZM241385>XAC>theophylline, as well as the agonist NECA. Furthermore, the receptor was made with its large (ca. 120 amino acid) C-terminal domain intact. dG hA2aR was purified to homogeneity in three steps, and its identity confirmed by electrospray tandem mass spectrometry following digestion with trypsin. The secondary structure of the entire receptor is largely (ca. 81%) alpha-helical. Purified dG hA2aR bound [(3)H]ZM241385 in a saturable manner with a B(max) of 18.1+/-0.5 nmol/mg protein, close to the theoretical B(max) value for pure protein (21.3 nmol/mg protein), showing that the receptor had retained its functionality during the purification process. Regular production of pure dG hA2aR in milligram quantities has enabled crystallisation trials to be started.
Assuntos
Expressão Gênica , Receptor A2A de Adenosina/isolamento & purificação , Receptor A2A de Adenosina/metabolismo , Membrana Celular/metabolismo , Dicroísmo Circular , Clonagem Molecular , Glicosilação , Humanos , Plasmídeos/genética , Estrutura Secundária de Proteína , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Solubilidade , Transformação Genética/genéticaRESUMO
The adenosine A(2A) receptor (A(2A)R) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A(2A)R gene expression, we have characterized the genomic organization of the mouse and human A(2A)R genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5' untranslated regions (5'-UTRs) of mouse A(2A)R mRNA were identified by rapid amplification of 5' cDNA end (5' RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding "exon 1" (h1A, h1B, h1C, h1D, h1E and h1F) in the human A(2A)R gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5' oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A(2A)R transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A(2A)R gene by 5' oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A(2A)R gene expression. Finally, our analysis indicates that A(2A)R mRNA and a novel transcript partially overlapping with the 3' exon h3, but in opposite orientation to the A(2A)R gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A(2A)R genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5'-UTR. This raises the possibility of generating multiple tissue-specific A(2A)R mRNA species by alternative promoters with varying regulatory susceptibility.