Functionality of a Membrane Protein in Bicelles

Czerski, Lech; Sanders, Charles R.

doi:10.1006/abio.2000.4720

Cited by 95 publications

(110 citation statements)

References 24 publications

(27 reference statements)

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“…This was reported by Sanders and Landis (11) for the ␣-helical P24 (K 2 GL 24 K 2 A), in which the 36 Å polyleucine hydrophobic stretch could not be incorporated into bicelles. Similarly, DAGK was shown to have a higher enzymatic activity in the thicker DPPC bicelles than in those containing DLPC or DMPC (21). However, bicelles can be made using phospholipids with longer acyl chains.…”

Section: Resultsmentioning

confidence: 96%

“…For the bicelles organized as disks or perforated lamellae, the planar DMPC section composing these structures is interesting for the study of peptides and proteins as it is similar to that of the biomembranes. Importantly, it has been shown that the biological activity of the enzyme diacylglycerolkinase (DAGK) is preserved in bicelles (11,21,82). In addition, these model membranes orient at physiological temperatures and, just like biomembranes, are in the fluid phase and contain phosphatidylcholines (28).…”

Section: (B)mentioning

confidence: 99%

“…Because the bicelle interior consists of a true lipid bilayer, this system presents a more natural environment for membrane proteins. It has been shown that the activity of some proteins and enzymes incorporated into micelles is not readily preserved (8 -10) but that the biological activity of the enzyme diacylglycerolkinase (DAGK) is preserved in bicelles (11,21,82). In addition, it has been shown that the position of a peptide in a micelle can be significantly different from its position in a phospholipid bilayer (108,109).…”

Section: Introductionmentioning

confidence: 99%

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Bicelles as model membranes for solid‐ and solution‐state NMR studies of membrane peptides and proteins

Marcotte

Auger

2005

Concepts Magnetic Resonance

189

196

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ABSTRACT:Bicelles are an attractive membrane mimetic system because of their planar surface and lipid composition, which resemble biological membranes. In addition, their orientation and morphologic properties make them amenable to solid-and solution-state NMR. This article reviews the physical properties of bicelles, such as magnetic alignment and viscosity as well as the different models proposed in the literature to explain the bicelle morphology. The utility of bicelles for studying the interaction and structure of membrane peptides and proteins by solid-and solution-state NMR is also presented, along with the advantages and limitations of bicelles.

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Section: Resultsmentioning

confidence: 96%

Section: (B)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bicelles as model membranes for solid‐ and solution‐state NMR studies of membrane peptides and proteins

Marcotte

Auger

2005

Concepts Magnetic Resonance

189

196

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“…The water-soluble DgkA substrate, ATP, and product, ADP, must, respectively, diffuse into and out of the mesophase in the course of the assay to support kinase activity and for detection. The latter is done in the current application using a coupled enzyme system (19)(20)(21) that includes pyruvate kinase (PK), lactate dehydrogenase (LD), phosphoenolpyruvate (PEP), and nicotinamide adenine dinucleotide (NADH). Thus, ADP production as a result of DgkA activity leads to a decrease in NADH concentration in the bathing aqueous solution that can be monitored conveniently by following the change in dinucleotide absorption at 340 nm.…”

mentioning

confidence: 99%

Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes

Caffrey

2011

Proc. Natl. Acad. Sci. U.S.A.

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The lipidic cubic mesophase has been used to crystallize important membrane proteins for high-resolution structure determination. To date, however, no integral membrane enzymes have yielded to this method, the in meso. For a crystal structure to be meaningful the target protein must be functional. Using the in meso method with a membrane enzyme requires that the protein is active in the mesophase that grows crystals. Because the cubic phase is sticky and viscous and is bicontinuous topologically, quantitatively assessing enzyme activity in meso is a challenge. Here, we describe a procedure for characterizing the catalytic properties of the integral membrane enzyme, diacylglycerol kinase, reconstituted into the bilayer of the lipidic cubic phase. The kinase activity of this elusive crystallographic target was monitored spectrophotometrically using a coupled assay in a high-throughput, 96-well plate format. In meso, the enzyme exhibits classic Michaelis-Menten kinetics and works with a range of lipid substrates. The fact that the enzyme and its lipid substrate and product remain confined to the porous mesophase while its water-soluble substrate and product are free to partition into the aqueous bathing solution suggests a general and convenient approach for characterizing membrane enzymes that function with lipids in a membrane-like environment. The distinctive rheology of the cubic phase means that a procedural step to physically separate substrate from product is not needed.Because of its open, bicontinuous nature, the cubic phase offers the added benefit that the protein is accessible for assay from both sides of the membrane.coupled enzyme assay | DgkA | glycerophospholipid | phosphatidylglycerol phosphate synthase | PgsA T he lipidic cubic phase has been used to grow crystals of membrane proteins for high-resolution structure determination. This approach has had spectacular successes of late with the publication of structures for G-protein coupled receptors (1-7). The method has also been used with a variety of other membrane protein and peptide types that include photo-sensitive proteins like the assorted bacterial rhodopsins, a light harvesting complex and the photosynthetic reaction centers, a transporter, a channel, and an adhesin (8-15). Conspicuous by their absence on this list are the integral membrane enzyme class of proteins.We have an interest in the structure-function relationships that pertain to membrane enzymes involved in glycerolipid metabolism. Our immediate focus is on the diacylglycerol kinase, DgkA, in Escherichia coli responsible for the ATP-dependent conversion of diacylglycerol to phosphatidic acid for use in membranederived oligosaccharide synthesis (16). A solution NMR structure of this small, hydrophobic trimer was published recently (17), and a high-resolution crystal structure is needed now to complement and extend the solution work. Efforts at crystallizing DgkA by conventional means have produced crystals but none of structure quality (18). The lipidic cubic mesophase (in meso) m...

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“…1,2 A nanometer-sized discoidal structure, nanodisc, consisting of lipids and membrane scaffold proteins, [3][4][5] and a disc-shaped lipid-detergent mixture, bicelle, [6][7][8] have been developed to stabilize membrane proteins. An alternative method is to use amphipathic polymers containing both of hydrophobic and hydrophilic groups.…”

Section: Introductionmentioning

confidence: 99%

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

Han

Lee

et al. 2014

Protein Science

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Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-c-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ET A ) in their active conformations. Furthermore, ET A in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.

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Functionality of a Membrane Protein in Bicelles

Cited by 95 publications

References 24 publications

Bicelles as model membranes for solid‐ and solution‐state NMR studies of membrane peptides and proteins

Bicelles as model membranes for solid‐ and solution‐state NMR studies of membrane peptides and proteins

Lipid cubic phase as a membrane mimetic for integral membrane protein enzymes

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

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