RESUMO
G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs: (1) cell-free in vitro translation, (2) HEK cells, and (3) Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration, and can be analyzed with standard biophysical techniques and biochemical assays.
Assuntos
Cromatografia de Afinidade , Expressão Gênica , Receptores Acoplados a Proteínas G , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Sistema Livre de Células , Escherichia coli , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
G protein-coupled receptors (GPCRs) are vital for diverse biological functions, including vision, smell, and aging. They are involved in a wide range of diseases, and are among the most important targets of medicinal drugs. Tools that facilitate GPCR studies or GPCR-based technologies or therapies are thus critical to develop. Here we report using our QTY (glutamine, threonine, tyrosine) code to systematically replace 29 membrane-facing leucine, isoleucine, valine, and phenylalanine residues in the transmembrane α-helices of the GPCR CXCR4. This variant, CXCR4QTY29, became more hydrophilic, while retaining the ability to bind its ligand CXCL12. When transfected into HEK293 cells, it inserted into the cell membrane, and initiated cellular signaling. This QTY code has the potential to improve GPCR and membrane protein studies by making it possible to design functional hydrophilic receptors. This tool can be applied to diverse α-helical membrane proteins, and may aid in the development of other applications, including clinical therapies.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Cromatografia de Afinidade , Dicroísmo Circular , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7. We show that QTY code-designed chemokine receptor variants retain their thermostabilities, α-helical structures, and ligand-binding activities in buffer and 50% human serum. CCR5QTY, CXCR4QTY, and CXCR7QTY also bind to HIV coat protein gp41-120. Despite substantial transmembrane domain changes, the detergent-free QTY variants maintain stable structures and retain their ligand-binding activities. We believe the QTY code will be useful for designing water-soluble variants of membrane proteins and other water-insoluble aggregated proteins.
Assuntos
Glutamina/metabolismo , Receptores de Quimiocinas/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Detergentes/química , Glutamina/química , Glutamina/genética , Temperatura Alta , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Ligação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Solubilidade , Treonina/química , Treonina/genética , Tirosina/química , Tirosina/genética , Água/químicaRESUMO
Insects transmit numerous devastating diseases, including malaria, dengue fever, and sleeping sickness. Olfactory cues guide insects to their hosts, and are thus responsible for disease transmission. Understanding the molecular basis of insect olfaction could facilitate the development of interventions. The first step is to heterologously overexpress and purify insect olfactory receptors (ORs). This is challenging, as ORs are membrane proteins. Here, we show that insect ORs and their co-receptor can be expressed in an E. coli cell-free system. After immunoaffinity chromatography, the ORs are ~95% pure, and up to 1 mg/10 ml reaction is obtained. Circular dichroism together with microscale thermophoresis indicate that each receptor is properly folded, and can bind its respective ligand. This is the first time insect ORs have been expressed in an E. coli system. The methods described here could facilitate future structure-function studies, which may aid in developments to alleviate the suffering of millions caused by insect-transmitted diseases.
Assuntos
Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/genética , Neurônios Receptores Olfatórios/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Receptores Odorantes/isolamento & purificação , Animais , Sistema Livre de Células , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Escherichia coli/genética , Expressão Gênica/genética , Ligantes , Ligação Proteica , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores Odorantes/biossíntese , Receptores Odorantes/genéticaRESUMO
G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs' structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the overexpression host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs by cell-free in vitro translation systems, HEK cells, and Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration and can be analyzed with standard biophysical techniques and biochemical assays.
Assuntos
Receptores Acoplados a Proteínas G/genética , Cromatografia em Gel , Bicamadas Lipídicas , Modelos Moleculares , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/isolamento & purificaçãoRESUMO
BACKGROUND: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. PRINCIPAL FINDINGS: We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L(-1) of culture from E. coli with specific activities of 1000 U (Uâ=â1 µmol hydrogen evolved mg(-1) min(-1)). SIGNIFICANCE: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.
Assuntos
Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Chlamydomonas reinhardtii/enzimologia , Endopeptidases/metabolismo , Escherichia coli/metabolismo , Fator Xa/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Hidrogenase/genética , Hidrogenase/isolamento & purificação , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/isolamento & purificação , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Two binder candidates 4-C37L34-B and 3-C15L8-B from a 16-membered set of 42-residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence-based screening assay. Two carbonic anhydrase isoforms with 60 % homology exist in human blood with HCAI being present in five- to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16-membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4-C37L34-B bound HCAII with a K(D) of 17 nM and HCAI with a K(D) of 470 nM, that is, with a 30-fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two carbonic anhydrases were 60 and 390 nM, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.
Assuntos
Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Cumarínicos/química , Peptídeos/genética , Sulfonamidas/química , Sequência de Aminoácidos , Cumarínicos/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Sulfonamidas/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
The structural heterogeneity and thermal denaturation of a dansyl-labeled four-helix bundle homodimeric peptide was studied with steady-state and time-resolved fluorescence spectroscopy and with circular dichroism (CD). At room temperature the fluorescence decay of the polarity-sensitive dansyl, located in the hydrophobic core region, can be described by a broad distribution of fluorescence lifetimes, reflecting the heterogeneous microenvironment. However, the lifetime distribution is nearly bimodal, which we ascribe to the presence of two major conformational subgroups. Since the fluorescence lifetime reflects the water content of the four-helix bundle conformations, we can use the lifetime analysis to monitor the change in hydration state of the hydrophobic core of the four-helix bundle. Increasing the temperature from 9 degrees C to 23 degrees C leads to an increased population of molten-globule-like conformations with a less ordered helical backbone structure. The fluorescence emission maximum remains constant in this temperature interval, and the hydrophobic core is not strongly affected. Above 30 degrees C the structural dynamics involve transient openings of the four-helix bundle structure, as evidenced by the emergence of a water-quenched component and less negative CD. Above 60 degrees C the homodimer starts to dissociate, as shown by the increasing loss of CD and narrow, short-lived fluorescence lifetime distributions.
Assuntos
Peptídeos/química , Água/química , Sequência de Aminoácidos , Dicroísmo Circular , Dimerização , Lisina , Dados de Sequência Molecular , Peptídeos/metabolismo , Probabilidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Temperatura , Fatores de TempoRESUMO
We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR-FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMAC) chips containing about 10 mum thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR-FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)-tagged synthetic peptides of 25 (25-His6) and 48 (48-His6) amino acids to the NTA-groups and 3) attachment of a ligand, mesyl amide, to the surface-bound 48-His6. Despite interference from H(2)O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of approximately 100 mg mL(-1) and a dissociation constant of 116+/-11 muM, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR-FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm(-1). Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand-alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set-ups.
RESUMO
Human glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl group from a solid support under conditions compatible with standard protein purification schemes. A number of fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity toward hGST A1-1 and the A216K mutant. Substitutions at the alpha-NH2 part of the glutathione backbone were not tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized in future protein purification and labeling experiments.
Assuntos
Corantes Fluorescentes/química , Glutationa Transferase/química , Lisina/química , Ésteres/química , Corantes Fluorescentes/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Compostos de Sulfidrila/químicaRESUMO
Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 x 10(7) M(-1). The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a k(cat) of 0.0032 min(-1) and a KM of 41 microM. The previously reported catalysis of GSB has a k(cat) of 0.00078 min(-1) and KM of 5 microM. The k(cat) for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (k(cat)/KM)/k(uncat) for GS-2 is 3 x 10(6) M(-1). The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.
Assuntos
Glutationa Transferase/genética , Isoenzimas/genética , Engenharia de Proteínas/métodos , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Catálise , Histidina/genética , Humanos , Especificidade por SubstratoRESUMO
Previously, we discovered that human glutathione transferase (hGST) A1-1 could be site-specifically acylated on a tyrosine residue (Y9) to form ester products using thiolesters of glutathione (GS-thiolesters) as acylating reagents. Out of a total of 20 GS-thiolester reagents tested, 15 (75%) are accepted by hGST A1-1 and thus this is a very versatile reaction. The present investigation was aimed at obtaining a more stable product, an amide bond, between the acyl group and the protein, in order to further increase the value of the reaction. Three lysine mutants (Y9K, A216K, and Y9F/A216K) were therefore prepared and screened against a panel of 18 GS-thiolesters. The Y9K mutant did not react with any of the reagents. The double mutant Y9F/A216K reacted with only one reagent, but in contrast, the A216K mutant could be acylated at the introduced lysine 216 with eight (44%) of the GS-thiolesters. The reaction can take place in the presence of glutathione and even in a crude cell lysate for five (28%) of the reagents. Through the screening process we obtained some basic rules relating to reagent requirements. We have thus produced a mutant (A216K) that can be rapidly and site-specifically modified at a lysine residue to form a stable amide linkage with a range of acyl groups. One of the successful reagents is a fluorophore that potentially can be used in downstream protein purification and protein fusion applications.
Assuntos
Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Lisina/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Humanos , Hidrólise , Isoenzimas/química , Isoenzimas/genética , Ligantes , Lisina/química , Mutagênese Sítio-Dirigida , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.