scholarly journals N-Terminal Fusion Tags for Effective Production of G-Protein-Coupled Receptors in Bacterial Cell-Free Systems

Acta Naturae ◽  
2012 ◽  
Vol 4 (4) ◽  
pp. 58-64 ◽  
Author(s):  
E. N. Lyukmanova ◽  
Z. O. Shenkarev ◽  
N. F. Khabibullina ◽  
D. S. Kulbatskiy ◽  
M. A. Shulepko ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Bacterial Cell ◽  
Somatostatin Receptor ◽  
Rnase A ◽  
G Protein Coupled Receptors ◽  
Functional Studies ◽  
Terminal Fragment ◽  
Low Efficiency ◽  
G Protein Coupled

G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts from E. coli cells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cellfree production of the human 2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin from Exiguobacterium sibiricum (ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein from B. subtilis allows to increase the CF synthesis of the target GPCRs by 538 times, resulting in yields of 0.63.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

scholarly journals Stabilization of Membrane Proteins: the Case of G‐Protein‐Coupled Receptors

2008 ◽  
Vol 8 (3) ◽  
pp. 207-217 ◽  
Author(s):  
A. Reyes‐Alcaraz ◽  
T. Tzanov ◽  
P. Garriga
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
G Protein Coupled

scholarly journals A Homogeneous Enzyme Fragment Complementation-Based β-Arrestin Translocation Assay for High-Throughput Screening of G-Protein-Coupled Receptors

2008 ◽  
Vol 13 (8) ◽  
pp. 737-747 ◽  
Author(s):  
Xiaoning Zhao ◽  
Adrie Jones ◽  
Keith R. Olson ◽  
Kun Peng ◽  
Tom Wehrman ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Somatostatin Receptor ◽  
Gene Families ◽  
G Protein Coupled Receptors ◽  
Functional Assay ◽  
Gpcr Activation ◽  
Fragment Complementation ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. ( Journal of Biomolecular Screening 2008:737-747)

scholarly journals Machine Learning for Prioritization of Thermostabilizing Mutations for G-protein Coupled Receptors

2019 ◽  
Author(s):  
S. Muk ◽  
S. Ghosh ◽  
S. Achuthan ◽  
X. Chen ◽  
X. Yao ◽  
...  
Keyword(s):  
Machine Learning ◽  
Membrane Proteins ◽  
G Protein ◽  
Three Dimensional ◽  
G Protein Coupled Receptors ◽  
Complement Factor ◽  
Computational Framework ◽  
Alanine Scanning ◽  
Structure And Dynamics ◽  
G Protein Coupled

AbstractAlthough the three-dimensional structures of G-protein-coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence, structure and dynamics based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available dataset. A blind prediction for thermostable mutations of the Complement factor C5a Receptor retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.Statement Of SignifiganceG-protein-coupled receptors (GPCRs), the largest superfamily of membrane proteins play a vital role in cellular physiology and are targets to blockbuster drugs. Hence it is imperative to solve the three dimensional structures of GPCRs in various conformational states with different types of ligands bound. To reduce the experimental burden in identifying thermostable GPCR mutants, we report a computational framework using machine learning algorithms trained on thermostability data for 1231 mutants and features calculated from analysis of GPCR sequences, structure and dynamics to predict thermostable mutations ahead of experiments. This work represents a significant advancement in the development, validation and testing of a computational framework that can be extended to other class A GPCRs and helical membrane proteins.

scholarly journals Development of OPLS-AA/M Parameters for Simulations of G Protein-Coupled Receptors and Other Membrane Proteins

2022 ◽  
Author(s):  
Michael J. Robertson ◽  
Georgios Skiniotis
Keyword(s):  
Membrane Proteins ◽  
Force Field ◽  
G Protein ◽  
Drug Targets ◽  
G Protein Coupled Receptors ◽  
Dynamic Nature ◽  
Post Translational Modifications ◽  
Dynamics Simulations ◽  
High Level ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) and other membrane proteins are valuable drug targets, and their dynamic nature makes them attractive systems for study with molecular dynamics simulations and free energy approaches. Here, we report the development, implementation, and validation of OPLS-AA/M force field parameters to enable simulations of these systems. These efforts include the introduction of post-translational modifications including lipidations and phosphorylation. We also modify previously reported parameters for lipids to be more consistent with the OPLS-AA force field standard and extend their coverage. These new parameters are validated on a variety of test systems, with the results compared to high-level quantum mechanics calculations, experimental data, and simulations with CHARMM36m where relevant. The results demonstrate that the new parameters reliably reproduce the behavior of membrane protein systems.

scholarly journals Lipid nanoparticle technologies for the study of G protein-coupled receptors in lipid environments

2020 ◽  
Vol 12 (6) ◽  
pp. 1287-1302 ◽  
Author(s):  
Steven Lavington ◽  
Anthony Watts
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Drug Targets ◽  
Large Family ◽  
G Protein Coupled Receptors ◽  
Integral Membrane Proteins ◽  
Lipid Nanoparticle ◽  
Wide Range ◽  
Biophysical Studies ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function.

scholarly journals Phosphatidic acid phospholipase A1 mediates ER–Golgi transit of a family of G protein–coupled receptors

2014 ◽  
Vol 206 (1) ◽  
pp. 79-95 ◽  
Author(s):  
Govind Kunduri ◽  
Changqing Yuan ◽  
Velayoudame Parthibane ◽  
Katherine M. Nyswaner ◽  
Ritu Kanwar ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Phosphatidic Acid ◽  
G Protein ◽  
Golgi Complex ◽  
G Protein Coupled Receptors ◽  
Phospholipase A1 ◽  
Copii Vesicles ◽  
G Protein Coupled

The coat protein II (COPII)–coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein–coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.

Synthetic GPCRs and signal transduction cascades

2019 ◽  
Vol 3 (5) ◽  
pp. 609-614 ◽  
Author(s):  
Colleen Mulvihill ◽  
Andrew Ellington
Keyword(s):  
Signal Transduction ◽  
Synthetic Biology ◽  
Membrane Proteins ◽  
G Protein ◽  
Drug Targets ◽  
G Protein Coupled Receptors ◽  
Complex Signal ◽  
Diverse Group ◽  
Orthogonal Components ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are a large and diverse group of membrane proteins that constitute over 30% of FDA approved drug targets. Despite their importance, much remains unknown about GPCR signaling at a system's level. Efforts to engineer receptors with orthogonal components have attempted to provide tools to parse signaling and resultant phenotypes. Recent advances in synthetic biology provide opportunities to engineer receptors at scale and with additional properties that could further inform GPCR biology at a system's level, and enhance the ability to engineer complex signal transduction.

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