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 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.

Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing

2015 ◽  
Vol 396 (9-10) ◽  
pp. 1097-1107 ◽  
Author(s):  
Helmut Merk ◽  
Ralf-Bernhardt Rues ◽  
Christine Gless ◽  
Kerstin Beyer ◽  
Fang Dong ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Insect Cell ◽  
G Protein Coupled Receptors ◽  
Cellular Systems ◽  
Membrane Translocation ◽  
Cell Lysates ◽  
Fab Fragments ◽  
G Protein Coupled

Abstract G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

scholarly journals Simple Genetic Selection Protocol for Isolation of Overexpressed Genes That Enhance Accumulation of Membrane-Integrated Human G Protein-Coupled Receptors in Escherichia coli

2010 ◽  
Vol 76 (17) ◽  
pp. 5852-5859 ◽  
Author(s):  
Georgios Skretas ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
G Protein ◽  
Protein Production ◽  
Genetic Selection ◽  
G Protein Coupled Receptors ◽  
Efficient Production ◽  
E Coli ◽  
G Protein Coupled

ABSTRACT The efficient production of membrane proteins in bacteria remains a major challenge. In this work, we sought to identify overexpressed genes that enhance the yields of recombinant membrane proteins in Escherichia coli. We developed a genetic selection system for bacterial membrane protein production, consisting of membrane protein fusions with the enzyme β-lactamase and facile selection of high-production strains on ampicillin-containing media. This system was used to screen the ASKA library, an ordered library of plasmids encoding all the known E. coli open reading frames (ORFs), and several clones with the ability to accumulate enhanced amounts of recombinant membrane proteins were selected. Notably, coexpression of ybaB, a gene encoding a putative DNA-binding protein of unknown function, was found to enhance the accumulation of a variety of membrane-integrated human G protein-coupled receptors and other integral membrane proteins in E. coli by up to 10-fold. The results of this study highlight the power of genetic approaches for identifying factors that impact membrane protein biogenesis and for generating engineered microbial hosts for membrane protein production.

Sign in / Sign up

Export Citation Format

Share Document