Development of a Thermofluor assay for stability determination of membrane proteins using the Na+/H+antiporter NhaA and cytochromecoxidase

Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochromecoxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.

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Faculty Opinions recommendation of The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733315065.793568683 ◽

2019 ◽

Author(s):

Koreaki Ito

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Protein Complex ◽

Membrane Protein Complex ◽

Er Membrane

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Ab-initio determination of the shape of membrane proteins in a nanodisc

10.1101/2020.09.11.293043 ◽

2020 ◽

Author(s):

Simone Orioli ◽

Carl G. Henning Hansen ◽

Lise Arleth

Keyword(s):

User Interface ◽

Membrane Proteins ◽

Membrane Protein ◽

Ab Initio ◽

Analytical Model ◽

Hybrid Approach ◽

Ab Initio Methods ◽

Saxs Intensity ◽

Computational Speed

AbstractWe introduce a new software, called Marbles, that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the nanodisc contribution to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as set of beads, similarly to well known ab-initio methods. The code, implemented in C++ with a Python user interface, provides a good performance and includes the possibility to systematically treat unstructured domains. We prove the reliability and flexibility of our approach by benchmarking the code on a toy model and two proteins of very different geometry and size.

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Co-translational biogenesis of lipid droplet integral membrane proteins

Journal of Cell Science ◽

10.1242/jcs.259220 ◽

2021 ◽

Author(s):

Pawel Leznicki ◽

Hayden O. Schneider ◽

Jada V. Harvey ◽

Wei Q. Shi ◽

Stephen High

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Neutral Lipids ◽

Storage Site ◽

Hairpin Loop ◽

Membrane Protein Complex ◽

Molecule Inhibitor ◽

Protein Biogenesis ◽

Intracellular Storage ◽

Loop Conformation

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. This route may even result in a transient exposure of the short N-termini of some LD membrane proteins to the ER lumen, followed by putative topological rearrangements that would enable their transmembrane segment to form a hairpin loop and N-termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

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Decision letter: The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins

10.7554/elife.37018.022 ◽

2018 ◽

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Protein Complex ◽

Membrane Protein Complex ◽

Er Membrane

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Co-translational biogenesis of lipid droplet integral membrane proteins

10.1101/2021.08.05.455205 ◽

2021 ◽

Author(s):

Pawel Leznicki ◽

Wei Q Shi ◽

Stephen High

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Neutral Lipids ◽

Storage Site ◽

Hairpin Loop ◽

Membrane Protein Complex ◽

Molecule Inhibitor ◽

Protein Biogenesis ◽

Intracellular Storage ◽

Loop Conformation

Membrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. Strikingly, this route can also result in a transient exposure of the short N-termini of LD membrane proteins to the ER lumen, followed by topological rearrangements that enable their transmembrane segment to form a hairpin loop and N-termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

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Ab initio determination of the shape of membrane proteins in a nanodisc

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320015405 ◽

2021 ◽

Vol 77 (2) ◽

pp. 176-193

Author(s):

Simone Orioli ◽

Carl G. Henning Hansen ◽

Lise Arleth

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Ab Initio ◽

Analytical Model ◽

Hybrid Approach ◽

Saxs Intensity ◽

Computational Speed

New software, called Marbles, is introduced that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the contribution of the nanodisc to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as a set of beads, similarly to as in well known ab initio methods. The reliability and flexibility of this approach is proved by benchmarking the code, implemented in C++ with a Python interface, on a toy model and two proteins with very different geometry and size.

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Membrane protein megahertz crystallography at the European XFEL

Nature Communications ◽

10.1038/s41467-019-12955-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 11

Author(s):

Chris Gisriel ◽

Jesse Coe ◽

Romain Letrun ◽

Oleksandr M. Yefanov ◽

Cesar Luna-Chavez ◽

...

Keyword(s):

Data Collection ◽

Membrane Protein ◽

Protein Complexes ◽

Membrane Protein Complex ◽

Unit Cell Size ◽

Data Collection And Analysis ◽

Rapid Pulse ◽

Membrane Protein Complexes ◽

Increasing Demand

Abstract The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.

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ER membrane protein complex is required for the insertions of late-synthesized transmembrane helices of Rh1 in Drosophila photoreceptors

Molecular Biology of the Cell ◽

10.1091/mbc.e19-08-0434 ◽

2019 ◽

Vol 30 (23) ◽

pp. 2890-2900 ◽

Cited By ~ 5

Author(s):

Naoki Hiramatsu ◽

Tatsuya Tago ◽

Takunori Satoh ◽

Akiko K. Satoh

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Protein Complex ◽

Model System ◽

Membrane Insertion ◽

Transmembrane Helices ◽

Membrane Protein Complex ◽

C Terminus ◽

Wide Range ◽

Er Membrane

Most membrane proteins are synthesized on and inserted into the membrane of the endoplasmic reticulum (ER), in eukaryote. The widely conserved ER membrane protein complex (EMC) facilitates the biogenesis of a wide range of membrane proteins. In this study, we investigated the EMC function using Drosophila photoreceptor as a model system. We found that the EMC was necessary only for the biogenesis of a subset of multipass membrane proteins such as rhodopsin (Rh1), TRP, TRPL, Csat, Cni, SERCA, and Na+K+ATPase α, but not for that of secretory or single-pass membrane proteins. Additionally, in EMC-deficient cells, Rh1 was translated to its C terminus but degraded independently from ER-associated degradation. Thus, EMC exerted its effect after translation but before or during the membrane integration of transmembrane domains (TMDs). Finally, we found that EMC was not required for the stable expression of the first three TMDs of Rh1 but was required for that of the fourth and fifth TMDs. Our results suggested that EMC is required for the ER membrane insertion of succeeding TMDs of multipass membrane proteins.

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