Gramicidin structure and disposition in highly curved membranes

Liu, W.; Caffrey, Martin

doi:10.1016/j.jsb.2004.12.007

Cited by 52 publications

(78 citation statements)

References 38 publications

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“…The quenching curve (Fig. 1) is analogous to that observed with other integral membrane proteins known to reconstitute in meso (13,15,26) and is convincing evidence that DgkA similarly reconstitutes into the bilayer of the cubic phase.…”

Section: Resultssupporting

confidence: 73%

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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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Section: Resultssupporting

confidence: 73%

“…Absorbance and fluorescence measurements on the protein in surfo and in meso were carried out as previously described (13,15,26) with minor modifications (see SI Text).…”

Section: Methodsmentioning

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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“…In this case any cleavage of ␣ENaC at an externally accessible cleavage site within the vicinity of the furin cleavage sites would cause the same channel activation. Such mechanisms of hydrophobic mismatch leading to effects on P o have been proposed to affect the gating of many ions channels including gramicidin (37)(38)(39)(40)(41)(42).…”

Section: Discussionmentioning

confidence: 99%

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Bengrine

Lis

et al. 2009

Journal of Biological Chemistry

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We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of ␣ENaC using the serine protease subtilisin. Trimeric channels formed with ␣FM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an ϳ10-fold decrease in amiloride-sensitive whole cell conductance as compared with ␣WT (2.2 versus 21.2 microsiemens ( S)). Both ␣WT and ␣FM were activated to similar levels by subtilisin cleavage. Channels formed with ␣FD, a construct that deleted the segment between the two furin sites (⌬175-204), exhibited an intermediate conductance of 13.2 S. More importantly, ␣FD retained the ability to be activated by subtilisin to 108.8 ؎ 20.9 S, a level not significantly different from that of subtilisin activated ␣WT (125.6 ؎ 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of ␣ was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with ␣LD, a construct that extended the deleted sequence of ␣FD by 17 amino acids (⌬175-221). Channels with ␣LD were uncleaved, exhibited low baseline activity (4.1 S), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex.It is well established that serine proteases activate the epithelial sodium channel (ENaC). 2 Activation occurs by direct mechanisms that induce channel subunit cleavage (1, 2) as well as those that are cleavage-independent but may involve cleavage of protease-activated membrane receptors (3). Channel cleavage studies have established that cellular proteases such as furin endogenously cleave the channel ␣ and ␥ subunits. Mutation of identified endogenous cleavage sites on both of these subunits diminished baseline activity, demonstrating a role for cleavage in ENaC activation.The acute effects of ENaC cleavage have largely relied on examining the effects of the protease trypsin on the ␣ and ␥ subunits. These studies have examined the effects of cleavage on wild type and furin cleavage-deficient ENaC in oocytes and epithelial cells (1,4,5). Although these have markedly improved our understanding of channel activation by serine proteases, they suffer from the main shortcoming that trypsin is a non-selective serine protease that can cleave after a single arginine residue (6, 7), and therefore, it only offers a limited tool for examining the mechanisms of cleavage at specific sites. Consistent with the reduced specificity for trypsin is the observation that ENaC retains its cleavage by this protease after mutation of consensus cleavage sites for furin (1).Despite their limitations, these studies have ind...

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“…Spectroscopic Measurements: UV-Visible Absorption: Spectra were recorded with a Uvikon XL dual beam spectrophotometer (Research Instruments International., San Diego, CA), as described (18). Data were collected in the range from 750 nm to 250 nm in 1 nm steps at 100 nm/min with air as the reference.…”

Section: Sample Preparationmentioning

confidence: 99%

“…A correction for this so-called inner filter effect has been applied to all relevant data following established procedures (19), as described (18).…”

Section: Sample Preparationmentioning

confidence: 99%

Interactions of Tryptophan, Tryptophan Peptides, and Tryptophan Alkyl Esters at Curved Membrane Interfaces

Liu

Caffrey

2006

Biochemistry

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Motivated by on-going efforts to understand the mechanism of membrane protein crystallogenesis and transport in the lipidic cubic phase, the nature of the interaction between tryptophan and the bilayer/aqueous interface of the cubic phase has been investigated. The association was quantified by partitioning measurements which enabled the free energy of interaction to be determined. Temperature-dependent partitioning was used to parse the association free energy change into its enthalpic and entropic components. As has been observed with tryptophan derivatives interacting with glycerophospholipid bilayers in vesicles, tryptophan partitioning in the cubic phase is enthalpy driven. This is in contrast to partitioning into apolar solvents which exhibits the classic hydrophobic effect whose hallmark is a favorable entropy change. These results with tryptophan are somewhat surprising given the simplicity, homogeneity and curvature of the interface that prevails in the case of the cubic phase. Nevertheless, the interaction between tryptophan and the mesophase is very slight as revealed by its low partition coefficient. Additional evidence in support of interaction was obtained by electronic absorption and fluorescence spectroscopy and fluorescence quenching. Partitioning proved insensitive to the lipid composition of the membrane, examined by doping with glycerophospholipids. However, the interaction could be manipulated in meaningful ways by the inclusion in the aqueous medium of salt, glycerol or urea. The effects seen with tryptophan were amplified rationally when measurements were repeated using tryptophan alkyl esters and with tryptophan peptides of increasing length. These findings are interpreted in the context of the insertion, folding and function of proteins in membranes.Keywords cubic phase; fluorescence quenching; lipid bilayers; membrane protein crystallization; small-angle X-ray diffraction Diesenhofer and Michel commented on the "unexpected" non-uniform distribution of tryptophan (W) in the L and M transmembranal subunits of the photosynthetic reaction center (1). Since this was the first membrane protein whose high-resolution, 3-dimensional structure †

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Gramicidin structure and disposition in highly curved membranes

Cited by 52 publications

References 38 publications

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

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

Alternative Mechanism of Activation of the Epithelial Na+ Channel by Cleavage

Interactions of Tryptophan, Tryptophan Peptides, and Tryptophan Alkyl Esters at Curved Membrane Interfaces

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