scholarly journals Understanding GPCR recognition and folding from NMR studies of fragments

RSC Advances ◽  
2018 ◽  
Vol 8 (18) ◽  
pp. 9858-9870 ◽  
Author(s):  
Jacopo Marino ◽  
Reto Walser ◽  
Martin Poms ◽  
Oliver Zerbe
Keyword(s):  
Protein Folding ◽  
Membrane Proteins ◽  
Membrane Insertion ◽  
Nmr Studies

Cotranslational protein folding is a vectorial process, and for membrane proteins, N-terminal helical segments are the first that become available for membrane insertion. Here fragments corresponding to these segments are investigated by NMR.

scholarly journals Insertion of plastidic β-barrel proteins into the outer envelopes of plastids involves an intermembrane space intermediate formed with Toc75-V/OEP80

2021 ◽  
Author(s):  
Lucia E Gross ◽  
Anna Klinger ◽  
Nicole Spies ◽  
Theresa Ernst ◽  
Nadine Flinner ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Import ◽  
Membrane Insertion ◽  
Intermembrane Space ◽  
Envelope Membrane ◽  
Outer Envelope ◽  
Pea Proteins ◽  
Outer Envelope Membrane ◽  
Correct Topology ◽  
Shed Light

Abstract The insertion of organellar membrane proteins with the correct topology requires the following: First, the proteins must contain topogenic signals for translocation across and insertion into the membrane. Second, proteinaceous complexes in the cytoplasm, membrane, and lumen of organelles are required to drive this process. Many complexes required for the intracellular distribution of membrane proteins have been described, but the signals and components required for the insertion of plastidic β-barrel-type proteins into the outer membrane are largely unknown. The discovery of common principles is difficult, as only a few plastidic β-barrel proteins exist. Here, we provide evidence that the plastidic outer envelope β-barrel proteins OEP21, OEP24, and OEP37 from pea (Pisum sativum) and Arabidopsis thaliana contain information defining the topology of the protein. The information required for translocation of pea proteins across the outer envelope membrane is present within the six N-terminal β-strands. This process requires the action of TOC (translocon of the outer chloroplast membrane). After translocation into the intermembrane space, β-barrel proteins interact with TOC75-V, as exemplified by OEP37 and P39, and are integrated into the membrane. The membrane insertion of plastidic β-barrel proteins is affected by mutation of the last β-strand, suggesting that this strand contributes to the insertion signal. These findings shed light on the elements and complexes involved in plastidic β-barrel protein import.

Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments

2019 ◽  
pp. 143-185 ◽  
Author(s):  
Erik Henrich ◽  
Frank Löhr ◽  
Julija Mezhyrova ◽  
Aisha Laguerre ◽  
Frank Bernhard ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Membrane Proteins ◽  
Solution Nmr ◽  
Nmr Studies

Lipid−Protein Nanodiscs as Reference Medium in Detergent Screening for High-Resolution NMR Studies of Integral Membrane Proteins

2010 ◽  
Vol 132 (16) ◽  
pp. 5628-5629 ◽  
Author(s):  
Zakhar O. Shenkarev ◽  
Ekaterina N. Lyukmanova ◽  
Alexander S. Paramonov ◽  
Lyudmila N. Shingarova ◽  
Vladimir V. Chupin ◽  
...  
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Integral Membrane Proteins ◽  
Nmr Studies ◽  
High Resolution Nmr ◽  
Reference Medium

scholarly journals Nanodisc, amphipol or detergent belts in cryoEM reconstructions of membrane proteins are similar and correspond to a common ordered solvent layer

2020 ◽  
Author(s):  
Veronica Zampieri ◽  
Alexia Gobet ◽  
Xavier Robert ◽  
Pierre Falson ◽  
Vincent Chaptal
Keyword(s):  
Aqueous Solution ◽  
Membrane Proteins ◽  
Membrane Insertion ◽  
3D Reconstructions ◽  
Density Distributions ◽  
Hydrophobic Patch ◽  
Different Shapes ◽  
Solvent Layer

SummaryTo maintain membrane proteins soluble in aqueous solution, amphipathic compounds are used to shield the hydrophobic patch of their membrane insertion, which forms a belt around the protein. This hydrophobic belt is seldom looked at due to the difficulty to visualize it. Cryo-EM is now offering this possibility, where belts are visible in 3D reconstructions. We investigated membrane proteins solved in nanodiscs, amphipols or detergents to analyze whether the nature of the amphipathic compound influences the belt size in 3D reconstructions. We identified belt boundaries in map-density distributions and measured distances for every reconstruction. We showed that all the belts create on average similar reconstructions, whether they originate from the same protein, or from protein from different shapes and structures. There is no difference among detergents or types of nanodisc used. These observations illustrate that the belt observed in 3D reconstructions corresponds to the minimum ordered layer around membrane proteins.

Functional diversity of three different DsbA proteins from Neisseria meningitidis

Microbiology ◽  
2004 ◽  
Vol 150 (9) ◽  
pp. 2993-3000 ◽  
Author(s):  
Sunita Sinha ◽  
Paul R. Langford ◽  
J. Simon Kroll
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Protein Folding ◽  
Membrane Proteins ◽  
Neisseria Meningitidis ◽  
Outer Membrane Proteins ◽  
Invasive Disease ◽  
Neisseria Lactamica ◽  
Neisseria Subflava ◽  
Neisseria Flavescens

The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes – nmb0278, nmb0294 and nmb0407 – encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.

scholarly journals Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 430
Author(s):  
Evan Van Aalst ◽  
Maryam Yekefallah ◽  
Anil K. Mehta ◽  
Isaac Eason ◽  
Benjamin Wylie
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Recombinant Expression ◽  
Protein Product ◽  
Inward Rectifier ◽  
Correlation Spectroscopy ◽  
Protein Yield ◽  
K Channel ◽  
Functional Assay ◽  
Nmr Studies

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.

scholarly journals 2P103 Direct monitoring of membrane protein folding process during in-vitro expression by Surface Enhanced IR spectroscopy(03. Membrane proteins,Poster)

2013 ◽  
Vol 53 (supplement1-2) ◽  
pp. S176
Author(s):  
Kenichi Ataka ◽  
Joachim Heberle ◽  
Axel Baumann ◽  
Silke Kerruth ◽  
Ramona Schlesinger ◽  
...  
Keyword(s):  
Protein Folding ◽  
Membrane Proteins ◽  
Ir Spectroscopy ◽  
Membrane Protein ◽  
In Vitro Expression ◽  
Folding Process ◽  
Membrane Protein Folding ◽  
Surface Enhanced
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