A YidC-like Protein in the Archaeal Plasma Membrane

Borowska, Marta; Dominik, Pawel K.; Anghel, S.; Kossiakoff, Anthony A.; Keenan, Robert J.

doi:10.1016/j.str.2015.06.025

Cited by 68 publications

(82 citation statements)

References 66 publications

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“…Mj 0480 has a relatively small solvent exposed surface area and was recalcitrant to crystallization even after extensive effort (Borowska et al, 2015). To evaluate whether sABs generated from the nanodisc library sorting could be used as crystallization chaperones for detergent-solubilized proteins, we attempted purification of eight sAB- Mj 0480 complexes in DDM by size exclusion chromatography.…”

Section: Resultsmentioning

confidence: 99%

“…After initial high-throughput screening, we obtained crystals for four sAB- Mj 0480 complexes in DDM. Optimization of one of the conditions (sAB M1- Mj 0480) (Figures 5A and 5B) ultimately yielded crystals that were solved to 3.5 Å resolution (Borowska et al, 2015). The other three conditions where there were crystal hits are being optimized.…”

Section: Resultsmentioning

confidence: 99%

“…N-terminal hexa-histidine constructs of Mj 0480 and two variants of T. maritima CorA (wild-type and D253K) were expressed and purified as previously described (Dalmas et al, 2010; Borowska et al, 2015). Biotinylated versions of Mj 0480 and CorA wild-type in detergent were prepared using 20- and 5-fold molar excess of EZ-Link-NHS-SS-PEG4-Biotin (Thermo), respectively in buffer containing 10 mM Hepes pH 7.5, 200 mM NaCl, and either 0.05% n-dodecyl-beta-D-maltoside (DDM, Affymetrix) or 0.1% Fos-Choline-12 (FC-12, Affymetrix) for Mj 0480, and 25 mM Hepes pH 7.5, 150 mM NaCl, 0.05% DDM, 20 mM MgCl 2 for CorA wild-type.…”

Section: Methodsmentioning

confidence: 99%

“…Synthetic antibody fragments (sABs) in pSFV4 or pRH2.2 vectors (kindly provided by SGC Toronto and S. Sidhu, respectively) were expressed and purified as described previously (Borowska et al, 2015) in two-step purification protocol on MabSelect SuRe Protein A column (GE Healthcare) and cation exchange Resource S 1 mL column (GE Healthcare), and were dialyzed into buffer A.…”

Section: Methodsmentioning

confidence: 99%

“…To form the protein-sAB complexes, Mj 0480 was incubated with saturating amount of sAB M1 and complex was purified and crystallized as previously described (Borowska et al, 2015). Complexes of CorA wild-type with sABs C10, C13 and C18 at 1:1.2 molar ratio were injected onto a Superdex200 10/300 GL column equilibrated with buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 0.025% DDM and either 1 mM EDTA or 20 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

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Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs

et al. 2016

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SUMMARY A major challenge in membrane biophysics is to define the mechanistic linkages between a protein’s conformational transitions and its function. We describe a novel approach to stabilize transient functional states of membrane proteins in native-like lipid environments allowing for their structural and biochemical characterization. This is accomplished by combining the power of antibody Fab-based phage display selection with the benefits of embedding membrane protein targets in lipid-filled nanodiscs. In addition to providing a stabilizing lipid environment, nanodiscs afford significant technical advantages over detergent-based formats. This enables the production of a rich pool of high performance Fab binders that can be used as crystallization chaperones, as fiducial markers for single particle cryo-EM and as probes of different conformational states. Moreover, nanodisc generated Fabs can be used to identify detergents that best mimic native membrane environments for use in biophysical studies.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs

et al. 2016

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Archaeal Cells

Chadha¹,

Seydel²,

Klingl³

2020

Encyclopedia of Life Sciences

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At a first glance, Archaea are quite similar to Bacteria on a structural level, and for a long time they were named ‘Archaebacteria’. They can form cocci, rods, spirals or irregular shaped cells and are equally sized as Bacteria. Together they are referred to as ‘Prokaryotes’ because neither Archaea nor Bacteria possess a nucleus. Although this term indeed might be helpful in habitual language use, it does not refer to a phylogenetic group. In fact, transcription and translation machineries of Archaea have much more in common with eukaryotic cells than with Bacteria. In addition, there are many features that remain characteristic for Archaea, given by the fact that many representatives live and thrive under extreme environmental conditions. Key Concepts By application of respective PCR techniques, Archaea can be found in almost every habitat and sometimes are even more abundant than bacteria. Archaea show special adaptations to their sometimes extreme environments, like caldarchaeols, which are more stable at high temperatures. Like Bacteria, Archaea are also surrounded by a lipid bilayer, but in the latter case, the lipid moiety consists of C 5 ‐isoprenoid units that are coupled to glycerol via ether bonds at (sn)‐2,3 positions of the glycerol. Cell walls can be as simple as a proteinaceous surface layer or as complicated as in some methanogens with multiple layers, additional sheaths enclosing several cells and even more complex cell wall compounds. Archaea exhibit a broad variety in cell appendages that are different from bacterial ones in fine structure, composition, biosynthesis and anchorage in the cell.

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Structural Basis of the Sec Translocon and YidC Revealed Through X-ray Crystallography

Tsukazaki

2019

Protein J

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Protein translocation and membrane integration are fundamental, conserved processes. After or during ribosomal protein synthesis, precursor proteins containing an N-terminal signal sequence are directed to a conserved membrane protein complex called the Sec translocon (also known as the Sec translocase) in the endoplasmic reticulum membrane in eukaryotic cells, or the cytoplasmic membrane in bacteria. The Sec translocon comprises the Sec61 complex in eukaryotic cells, or the SecY complex in bacteria, and mediates translocation of substrate proteins across/into the membrane. Several membrane proteins are associated with the Sec translocon. In Escherichia coli, the membrane protein YidC functions not only as a chaperone for membrane protein biogenesis along with the Sec translocon, but also as an independent membrane protein insertase. To understand the molecular mechanism underlying these dynamic processes at the membrane, high-resolution structural models of these proteins are needed. This review focuses on X-ray crystallographic analyses of the Sec translocon and YidC and discusses the structural basis for protein translocation and integration.

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A YidC-like Protein in the Archaeal Plasma Membrane

Cited by 68 publications

References 66 publications

Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs

Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs

Archaeal Cells

Structural Basis of the Sec Translocon and YidC Revealed Through X-ray Crystallography

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