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![Conjugation of empty OTG micelles. (A) Light microscope images of hanging drops containing OTG (30 mM), the amphiphilic chelator, Dinonyl (1 mM) and cations as indicated (3 mM). After 1 h of incubation at 19 °C in the presence of 200 mM NaCl, phase separation of oil-rich globules is observed. The red color in the presence of Fe 2+ derives from the [(Dinonyl) 3 :Fe 2+ ] associate. Scale bar in all images represents 100 μm. (B) All conditions as in (A), except that no chelator is present.](publication/343126844/figure/fig2/AS:916028171247616@1595409543291/Conjugation-of-empty-OTG-micelles-A-Light-microscope-images-of-hanging-drops.jpg)
Conjugation of empty OTG micelles. (A) Light microscope images of hanging drops containing OTG (30 mM), the amphiphilic chelator, Dinonyl (1 mM) and cations as indicated (3 mM). After 1 h of incubation at 19 °C in the presence of 200 mM NaCl, phase separation of oil-rich globules is observed. The red color in the presence of Fe 2+ derives from the [(Dinonyl) 3 :Fe 2+ ] associate. Scale bar in all images represents 100 μm. (B) All conditions as in (A), except that no chelator is present.
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![Figure 3. Role of the amphiphilic [(Dinonyl) 3 :Zn 2+ ] complex in...](publication/343126844/figure/fig3/AS:916028171223042@1595409543339/Role-of-the-amphiphilic-Dinonyl-3-Zn-2-complex-in-promoting-crystallization-of-bR_Q320.jpg)
![Figure 4. Crystallization of bR via [(Dinonyl) 3 :Ni 2+ ] conjugation....](publication/343126844/figure/fig4/AS:916028171247618@1595409543379/Crystallization-of-bR-via-Dinonyl-3-Ni-2-conjugation-Light-microscope-images-of_Q320.jpg)
A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringin...
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Context 1
... (OTG) micelles. Non-ionic β-d-1-thioglucoside (OTG) micelles, devoid of MPs, were conjugated via the amphiphilic chelator 4,4′-dinonyl-2,2′-dipyridyl (Dinonyl) in association with divalent metal cations: Zn 2+ , Fe 2+ , or Ni 2+ . Each cation is able to bind Dinonyl at a molar ratio of 1:3, respectively [(Dinonyl) 3 :M 2+ ] 24 (Fig. 2). Micellar conjugation was indeed found to be efficient with each of the three cations studied and led within 60 min to macroscopic phase separation in the form of oil-rich globules ( Fig. 2A). In the absence of the cations, the chelators precipitated as elongated www.nature.com/scientificreports/ threads ( Fig. 2A, left) and in the ...
Context 2
... with divalent metal cations: Zn 2+ , Fe 2+ , or Ni 2+ . Each cation is able to bind Dinonyl at a molar ratio of 1:3, respectively [(Dinonyl) 3 :M 2+ ] 24 (Fig. 2). Micellar conjugation was indeed found to be efficient with each of the three cations studied and led within 60 min to macroscopic phase separation in the form of oil-rich globules ( Fig. 2A). In the absence of the cations, the chelators precipitated as elongated www.nature.com/scientificreports/ threads ( Fig. 2A, left) and in the absence of the Dinonyl chelator, but retaining the same cation concentration, no phase separation was observed (Fig. ...
Context 3
... [(Dinonyl) 3 :M 2+ ] 24 (Fig. 2). Micellar conjugation was indeed found to be efficient with each of the three cations studied and led within 60 min to macroscopic phase separation in the form of oil-rich globules ( Fig. 2A). In the absence of the cations, the chelators precipitated as elongated www.nature.com/scientificreports/ threads ( Fig. 2A, left) and in the absence of the Dinonyl chelator, but retaining the same cation concentration, no phase separation was observed (Fig. ...
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... led within 60 min to macroscopic phase separation in the form of oil-rich globules ( Fig. 2A). In the absence of the cations, the chelators precipitated as elongated www.nature.com/scientificreports/ threads ( Fig. 2A, left) and in the absence of the Dinonyl chelator, but retaining the same cation concentration, no phase separation was observed (Fig. ...
Context 5
... PDCs had been obtained by dissociating purple membranes with OTG (see "Methods" section) and validating that the characteristic bR absorption spectrum had been preserved (online Supplementary Information, Figure S1). With the availability of micellar preparations containing native bR, the effects of three divalent cations (Fe 2+ , Co 2+ , Cd 2+ ) on the efficiency of conjugation were studied (online Supplementary Information, Figure S2). We found that the [(Dinonyl) 3 :Zn 2+ ] complex successfully conjugated micellar aggregates and generated large purple oil-rich globules (20-100 µm) after 1 day of incubation in the dark at 19 °C (Fig. 3A). ...
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... absorption spectrum of bR extracted from its native purple membrane with OTG (as described above) was measured ( Figure S6, A). The characteristic absorption of native bR at 565-568 nm was preserved when the amphiphilic chelator (Dinonyl), the metal (Ni 2+ ) or both were added and incubated for up to 24 h at 19 °C in the dark ( Figure S2, A). However, a decrease of ~ 20% in the OD of bR at 565-568 nm was observed after 1 day of incubation in the presence of the [(Dinonyl) 3 :Ni 2+ ] amphiphilic complex, presumably due to the ability of the latter to trigger bR aggregation. ...
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Citations
... Strong metal chelators such as 1,10-phenanthroline or bipyridine analogs have been tested, both in the absence and presence of MPs. However, the protein crystals generated were either too small or too thin to give useful X-ray diffraction, perhaps due to the overriding strength of the intermicellar interactions 20 . ...
... S1). No precipitant was required for micron-scale, dense aggregates to form; however, the membrane protein crystals generated were either too small or too thin to provide useful X-ray 20 . In the current work, we sought to move closer to our goal of fine-tuning the intermicellar interactions. ...
Specific conjugation of decyl -D-maltoside (DM) or dodecyl-D-maltoside (DDM) detergent micelles is accomplished between pH 7.0-8.5 in the presence of an amphiphilic analog of the amino acid histidine, bound to a 10-carbon hydrocarbon chain (His1-C10) and Ni2+ ions. Following addition of 10-15 wt% PEG-6000 as precipitant, phase separation in the form of oil-rich globules (30-600 µm) is observed by light microscopy. Other divalent cations: Zn2+, Fe2+, Cu2+ lead to dark precipitates rather than colorless globules; while Mg2+, Ca2+ do not promote any phase separation at all. Even in the absence of precipitant, dynamic light scattering (DLS) measurements demonstrate that DM micelles (hydrodynamic size ~ 6 nm) or DDM micelles (8 nm) self-associate into larger particles (9 nm and 411 nm for DM; 10 nm and 982 nm for DDM) in the presence of His1-C10 and nickel ions. Micellar conjugation is partially reversible in the presence of water soluble 50 mM EDTA, histidine or imidazole chelators. Cryo-transmission electron microscopy (cryo-TEM) imaging revealed the formation of non-uniformly dense detergent aggregates for both DM and DDM micelles in the presence of precipitant. The possible utility of such His1-tagged DM or DDM micelles for promoting crystallization of integral membrane proteins is discussed.
... However, the protein crystals generated were either too small or too thin, perhaps due to the overriding strength of the intermicellar interactions. 17 In the present work, we describe speci c conjugation of decyl-β-D-maltoside or dodecyl-β-D-maltoside micelles, accomplished at 19°C, pH 7.0-8.5 via a purpose synthesized, amphiphilic analog of the amino acid histidine, bound to a 10-carbon hydrocarbon chain (termed His 1 -C10; Fig. 1), which is able to chelate Ni 2+ ions (Fig. 2). We suggest that the partial reversibility of micelle conjugation observed in the presence of competing, water soluble metal chelators, may potentially provide su cient exibility to allow ne tuning and to promote stable crystal nucleation. ...
... His 1 -C10 / Ni 2+ complexes conjugate DM or DDM micelles In previous reports, [15][16][17]19 we have shown that strongly interacting amphiphilic [metal chelator:divalent cation] complexes, such as [1,10-phenanthroline or bipyridine analogs:Fe 2+ ] are able to conjugate DM or DDM micelles, both in the absence and presence of integral membrane proteins. The dissociation constant for Fe 2+ bound to 1,10-phenanthroline is K d ~10 − 21 M. [20][21] As such, the conjugated DM or DDM micellar aggregates were very condensed (Supplementary- Figure S1). ...
... No precipitant was required for micron-scale, dense aggregates to form; however, the membrane protein crystals generated were either too small or too thin to provide useful X-ray diffraction data. 17 In the current work, we sought to move closer to our goal of ne-tuning the inter-micellar interactions. Only in that way would the necessary conformational exibility be provided for stable crystal nuclei to form. ...
Specific conjugation of decyl β-D-maltoside (DM) or dodecyl β-D-maltoside (DDM) detergent micelles is accomplished between pH 7.0-8.5 in the presence of an amphiphilic analog of the amino acid histidine, bound to a 10-carbon hydrocarbon chain (His 1 -C10) and Ni ²⁺ ions. Following addition of 10–15 wt% PEG-6000 as precipitant, phase separation in the form of oil-rich globules (30–600 µm) is observed by light microscopy. Other divalent cations: Zn ²⁺ , Fe ²⁺ , Cu ²⁺ lead to dark precipitates rather than colorless globules; while Mg ²⁺ , Ca ²⁺ do not promote any phase separation at all. Even in the absence of precipitant, dynamic light scattering (DLS) measurements demonstrate that DM micelles (hydrodynamic size ~ 6 nm) or DDM micelles (8 nm) self-associate into larger particles (9 nm and 411 nm for DM; 10 nm and 982 nm for DDM) in the presence of His 1 -C10 and nickel ions. Micellar conjugation is partially reversible in the presence of water soluble 50 mM EDTA, histidine or imidazole chelators. Cryo-transmission electron microscopy (cryo-TEM) imaging revealed the formation of non-uniformly dense detergent aggregates for both DM and DDM micelles in the presence of precipitant. The possible utility of such His 1 -tagged DM or DDM micelles for promoting crystallization of integral membrane proteins is discussed.
... bR was extracted from purple membranes with OTG, as described previously. 18 To freshly extracted bR (10.5 μL of 3−5 mg/mL bR), 0.5 μL of 27 mM DP-nonyl or DPtert-butyl or DP-methyl (in EtOH) was added with vigorous stirring (for 20 s) and followed by the addition of 2.5 μL of 5.4 mM NiCl 2 (in DDW). Aliquots (4 μL) from the latter mixture were transferred to siliconized coverslides and incubated overnight at 19°C in the dark against a reservoir (0.5 mL) containing 0.5 M NaCl in VDX crystallization plates (from Hampton Research). ...
In the decades'-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein aggregation in aqueous solution and/or as a conformation stabilizing environment. We have focused on exploiting the physical chemistry of detergent micelles in order to direct intrinsic MP/detergent complexes to assemble via conjugation under ambient conditions, thereby permitting finely tuned control over the micelle cloud point. In the current work, three commercially available amphiphilic, bipyridine chelators in combination with Fe2+ or Ni2+ were tested for their ability to conjugate non-ionic detergent micelles both in the presence and absence of an encapsulated bacteriorhodopsin molecule. Water-soluble chelators were added, and results were monitored with light microscopy and dynamic light scattering (DLS). [Bipyridine:metal] complexes produced micellar conjugates, which appeared as oil-rich globules (10-200 μm) under a light microscope. DLS analysis demonstrated that micellar conjugation is complete 20 min after the introduction of the amphiphilic complex, and that the conjugation process can be fully or partially reversed with water-soluble chelators. This process of controlled conjugation/deconjugation under nondenaturing conditions provides broader flexibility in the choice of detergent for intrinsic MP purification and conformational flexibility during the crystallization procedure.
Membrane protein (MP) functional and structural characterization requires large quantities of high-purity protein for downstream studies. Barriers to MP characterization include ample overexpression, solubilization, and purification of target proteins while maintaining native activity and structure. These barriers can be overcome by utilizing an efficient purification protocol in a high-yield eukaryotic expression system such as Saccharomyces cerevisiae. S. cerevisiae offers improved protein folding and posttranslational modifications compared to prokaryotic expression systems. This chapter contains practices used to overcome barriers of solubilization and purification using S. cerevisiae that are broadly applicable to diverse membrane associated, and membrane integrated, protein targets.Key wordsMembrane proteinPurification
Saccharomyces cerevisiae
ChromatographyProtein production
