scholarly journals Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

2021 ◽  
Vol 22 (16) ◽  
pp. 8647
Author(s):  
Vanessa Boritzki ◽  
Harald Hübner ◽  
Anni Allikalt ◽  
Peter Gmeiner ◽  
Birgitta M. Wöhrl
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Dopamine Receptors ◽  
Drug Targets ◽  
Radioligand Binding ◽  
Receptor Subtype ◽  
Binding Assays ◽  
Intracellular Loop ◽  
Whole Cells ◽  
Affinity Ligand

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.

scholarly journals Identification of Discrete Domains within Gonococcal Transferrin-Binding Protein A That Are Necessary for Ligand Binding and Iron Uptake Functions

2000 ◽  
Vol 68 (12) ◽  
pp. 6988-6996 ◽  
Author(s):  
Ian C. Boulton ◽  
Mary Kate Yost ◽  
James E. Anderson ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Ligand Binding ◽  
Iron Uptake ◽  
Binding Protein ◽  
Protein A ◽  
Ligand Interaction ◽  
Whole Cells ◽  
Affinity Ligand ◽  
Discrete Domains ◽  
Transferrin Binding Proteins

ABSTRACT The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenicNeisseria spp. can sequester iron from this protein, dependent upon two iron-repressible, transferrin-binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a β-barrel exposing multiple surface loops, some of which are likely to contain ligand-binding motifs. In this study we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putative loops (designated loops 4, 5, and 8). Each mutant TbpA could be expressed without toxicity and was surface exposed as assessed by immunoblotting, transferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase assays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of the three mutated TbpAs were incapable of growth on transferrin as a sole iron source. These data implicate putative loops 4 and 5 as critical determinants for receptor function and transferrin-iron uptake by gonococcal TbpA. The phenotype of the ΔL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.

Differential development of umbilical and systemic arteries. I. ANG II receptor subtype expression

1998 ◽  
Vol 274 (3) ◽  
pp. R797-R807 ◽  
Author(s):  
Jeffrey R. Kaiser ◽  
Blair E. Cox ◽  
Timothy A. Roy ◽  
Charles R. Rosenfeld
Keyword(s):  
Blood Flow ◽  
Radioligand Binding ◽  
Plasma Renin ◽  
Receptor Subtype ◽  
Ang Ii ◽  
Binding Assays ◽  
Fetal Sheep ◽  
Umbilical Blood ◽  
Steady State Responses ◽  
Umbilical Arteries

In fetal sheep umbilical responses to angiotensin II (ANG II) exceed those by systemic vasculature. Two ANG II receptors (AT) exist, AT1 and AT2, but only AT1 mediates vasoconstriction in adult tissues. Thus differences in reactivity could reflect differences in subtype expression. Using competitive radioligand binding assays, we demonstrated AT1 predominance in umbilical arteries and AT2 in femoral arteries. Steady-state responses to intravenous ANG II (0.229–1.72 μg/min) were studied in 16 fetuses with umbilical and/or femoral artery flow probes without and with local AT1 (L-158,809) or AT2 (PD-123319) blockade. ANG II dose dependently ( P < 0.001) increased umbilical resistance more than arterial pressure (MAP) while decreasing umbilical blood flow. Femoral vascular resistance also increased dose dependently ( P = 0.02), but responses were less than umbilical ( P = 0.0001) and paralleled increases in MAP; blood flow was unaffected. Cumulative local doses of L-158,809 (125 μg) inhibited all responses ( P< 0.001); however, 1,000 μg of the AT2 antagonist had no effect. Plasma renin activity (PRA) was unaltered by local AT1 blockade, whereas PRA doubled ( P = 0.001) after systemic infusion of only 50 μg of the AT1 antagonist and remained elevated. Differences in umbilical and femoral vascular responses to ANG II are in large part due to differences in AT subtype expression. Furthermore, in fetal sheep the ANG II negative feedback on PRA is mediated by AT1 receptors, and it is substantially more sensitive to receptor blockade than the vasculature.

Neurotensin receptor type 1: Escherichia coli expression, purification, characterization and biophysical study

2007 ◽  
Vol 35 (4) ◽  
pp. 760-763 ◽  
Author(s):  
P.J. Harding ◽  
H. Attrill ◽  
S. Ross ◽  
J.R. Koeppe ◽  
A.N. Kapanidis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Single Molecule ◽  
Fluorescent Protein ◽  
Radioligand Binding ◽  
Yellow Fluorescent Protein ◽  
Active Form ◽  
X Ray Crystallography ◽  
Biophysical Study ◽  
Escherichia Coli Expression

NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.

scholarly journals High-Throughput Screening for Norepinephrine Transporter Inhibitors Using the FLIPRTetra

2007 ◽  
Vol 12 (3) ◽  
pp. 436-441 ◽  
Author(s):  
Ryan Wagstaff ◽  
Michael Hedrick ◽  
Jun Fan ◽  
Paul D. Crowe ◽  
Daniel DiSepio
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Radioligand Binding ◽  
Norepinephrine Transporter ◽  
Functional Screening ◽  
Monoamine Transporters ◽  
Binding Assays ◽  
Screening Assay ◽  
Fluorescent Substrate

Monoamine transporters regulate the concentration of neurotransmitters in the synapse following neurotransmission and are very important drug targets in the pharmaceutical industry. Because of the labor-intensive nature of functional uptake assays using radioactive substrates, high-throughput screening for monoamine transporter inhibitors has been limited to radioligand binding assays. In this article, the authors describe the development of a 384-well, high-throughput functional screening assay for norepinephrine transporter inhibitors using the FLIPRTetra and a recently identified fluorescent substrate, 4-(4-dimethylaminostyryl)- N-methyl-pyridinium (ASP+). ( Journal of Biomolecular Screening 2007:436-441)

scholarly journals The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kenichi Kamata ◽  
Kenji Mizutani ◽  
Katsuya Takahashi ◽  
Roberta Marchetti ◽  
Alba Silipo ◽  
...  
Keyword(s):  
Immune System ◽  
Sialic Acid ◽  
Ligand Binding ◽  
Steric Hindrance ◽  
Sequence Similarity ◽  
Binding Assays ◽  
Subunit Interface ◽  
Nmr Techniques ◽  
And Control ◽  
Asialo Gm1

AbstractSeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac$$\alpha$$ α (2-3)Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc) and its precursor, asialo-GM1 (Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the $$\beta$$ β -trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

scholarly journals Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xuesong Wang ◽  
Willem Jespers ◽  
Rubén Prieto-Díaz ◽  
Maria Majellaro ◽  
Adriaan P. IJzerman ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Structural Data ◽  
Binding Mode ◽  
Selective Recognition ◽  
Chiral Hplc ◽  
Binding Assays ◽  
Structural Determinants ◽  
X Ray Crystallography ◽  
Adenosine Receptor Antagonist ◽  
Energy Perturbation

AbstractThe four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

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