Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs

Ritchie, Tasha K.; Grinkova, Yelena V.; Bayburt, Timothy H.; Denisov, Ilia G.; Zolnerciks, Joseph K.; Atkins, William M.; Sligar, Stephen G.

doi:10.1016/s0076-6879(09)64011-8

Cited by 800 publications

(897 citation statements)

References 69 publications

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“…To avoid this problem, we prepared both cryoEM and negatively stained specimens immediately after the final step of purification. The apparent yield of PA-nanodisc complexes is substantially higher than in reconstitutions of other membrane proteins with detergent dialysis alone 11 ; we estimate that 40-50% of the nanodiscs in the final purification contain one or more PA, judged by EM of negatively-stained samples. 7 The presence of ''empty'' nanodiscs was unanticipated, given the extensive wash steps of the immobilized complexes in the first stage of purification.…”

Section: Resultsmentioning

confidence: 76%

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

et al. 2013

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We have visualized by cryo-electron microscopy (cryo-EM) the complex of the anthrax protective antigen (PA) translocon and the N-terminal domain of anthrax lethal factor (LF N ) inserted into a nanodisc model lipid bilayer. We have determined the structure of this complex at a nominal resolution of 16 Å by single-particle analysis and three-dimensional reconstruction. Consistent with our previous analysis of negatively stained unliganded PA, the translocon comprises a globular structure (cap) separated from the nanodisc bilayer by a narrow stalk that terminates in a transmembrane channel (incompletely distinguished in this reconstruction). The globular cap is larger than the unliganded PA pore, probably due to distortions introduced in the previous negatively stained structures. The cap exhibits larger, more distinct radial protrusions, previously identified with PA domain three, fitted by elements of the NMFF PA prepore crystal structure. The presence of LF N , though not distinguished due to the seven-fold averaging used in the reconstruction, contributes to the distinct protrusions on the cap rim volume distal to the membrane. Furthermore, the lumen of the cap region is less resolved than the unliganded negatively stained PA, due to the low contrast obtained in our images of this specimen. Presence of the LF N extended helix and N terminal unstructured regions may also contribute to this additional internal density within the interior of the cap. Initial NMFF fitting of the cryoEM-defined PA pore cap region positions the Phe clamp region of the PA pore translocon directly above an internal vestibule, consistent with its role in toxin translocation.

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Section: Resultsmentioning

confidence: 76%

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

et al. 2013

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“…Phospholipid bilayer nanodiscs were assembled essentially as previously described 45, incorporating CPR or CYP3A4 either singly, or together as described by Denisov et al . 42.…”

Section: Resultsmentioning

confidence: 99%

“…The temperature of nanodisc assembly is recommended to correspond to the melting temperature of the lipid used 45; for liver and mixed lipids, we expected this to be 20 °C or above. A series of trials were performed (with no inserted membrane protein) to compare different ratios of lipid to MSP at different temperatures.…”

Section: Resultsmentioning

confidence: 99%

“…MSPs were expressed and purified as described by Ritchie et al . 45, except that expression was induced in batch cultures of 0.5 L Terrific Broth (TB) in 2‐L flasks shaken at 30 °C and 200 r.p.m. for 5–6 h after induction.…”

Section: Methodsmentioning

confidence: 99%

“…Assembly of phospholipid bilayer nanodiscs was essential as previously described 42, 45. To constitute CPR into nanodiscs, CPR was mixed with the membrane scaffold protein MSP1D1 and cholate‐solubilized POPC at a molar ratio of 1 CPR: 65 MSP1D1: 130 POPC in a solution containing 10 m m POPC, 20 m m sodium cholate, 0.1 m NaCl, 20 m m Tris‐HCl, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

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Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase‐cytochrome P450 3A4 in nanodiscs

et al. 2017

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The haem‐containing mono‐oxygenase cytochrome P450 3A4 (CYP3A4) and its redox partner NADPH‐dependent cytochrome P450 oxidoreductase (CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane‐bound in the endoplasmic reticulum (ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady‐state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7‐benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono‐oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4‐nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR‐CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR‐CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity.

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The Use of Detergents to Purify Membrane Proteins

2016

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Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy. A brief summary of alternative membrane protein solubilizing and stabilizing systems is also included. Protocols in this unit include the isolation and solubilization of biological membranes and phase separation; support protocols for detergent removal, detergent exchange, and the determination of critical micelle concentration using different methods are also included.

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Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs

Cited by 800 publications

References 69 publications

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

Three dimensional structure of the anthrax toxin translocon–lethal factor complex by cryo‐electron microscopy

Liver microsomal lipid enhances the activity and redox coupling of colocalized cytochrome P450 reductase‐cytochrome P450 3A4 in nanodiscs

The Use of Detergents to Purify Membrane Proteins

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