scholarly journals Asymmetric maltose neopentyl glycol amphiphiles for a membrane protein study: effect of detergent asymmetricity on protein stability

2019 ◽  
Vol 10 (4) ◽  
pp. 1107-1116 ◽  
Author(s):  
Hyoung Eun Bae ◽  
Yang Du ◽  
Parameswaran Hariharan ◽  
Jonas S. Mortensen ◽  
Kaavya K. Kumar ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Stability ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Neopentyl Glycol ◽  
G Protein Coupled ◽  
Enhanced Stability

An asymmetric MNG, MNG-8,12, provided enhanced stability to human G protein-coupled receptors (GPCRs) compared to the symmetric MNG, MNG-3.

scholarly journals The G protein coupled receptor CXCR4 designed by the QTY code becomes more hydrophilic and retains cell signaling activity

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lotta Tegler ◽  
Karolina Corin ◽  
Horst Pick ◽  
Jennifer Brookes ◽  
Michael Skuhersky ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Membrane Protein ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Hek293 Cells ◽  
Biological Functions ◽  
Wide Range ◽  
Medicinal Drugs ◽  
G Protein Coupled ◽  
Signaling Activity

AbstractG 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.

scholarly journals Multiplexed analysis of the secretin-like GPCR-RAMP interactome

2019 ◽  
Vol 5 (9) ◽  
pp. eaaw2778 ◽  
Author(s):  
Emily Lorenzen ◽  
Tea Dodig-Crnković ◽  
Ilana B. Kotliar ◽  
Elisa Pin ◽  
Emilie Ceraudo ◽  
...  
Keyword(s):  
Membrane Protein ◽  
G Protein ◽  
Chemokine Receptors ◽  
G Protein Coupled Receptors ◽  
Orphan Receptors ◽  
Native Proteins ◽  
Receptor Activity Modifying Proteins ◽  
G Protein Coupled ◽  
Suspension Bead Array ◽  
Multiplexed Analysis

Receptor activity–modifying proteins (RAMPs) have been shown to modulate the functions of several G protein–coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

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.

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