Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Landau, Ehudâ€‰M.; Rosenbusch, J P

doi:10.1073/pnas.93.25.14532

Cited by 926 publications

(689 citation statements)

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“…The current study that relates to W/membrane interactions was motivated in part by our interest in understanding the mechanism by which membrane proteins crystallize in the lipidic cubic mesophase (9) by the so-called in meso method (reviewed in (10)). Crystals so produced are used for structure determination by crystallographic means.…”

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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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confidence: 99%

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

Liu

Caffrey

2006

Biochemistry

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“…(5) indicates that the centrifugal force acting on any microcapsule of the same row is identical. However, the centrifugal forces acting on different rows are different because of the variation of the distance from the center of the disc, which may affect the uniformity of the mixing.…”

Section: Design Of the Single Microcapsule For Mixingmentioning

confidence: 99%

“…In the in meso method, an extended bilayer composed of lipid, Keywords: highly viscous biosample, mixing, microcapsule, microchannel, centrifugation, cubic phase detergent, and target protein is presumed to form. It constitutes the following two steps for growing crystals: 4,5 (1) mix two parts protein solution/dispersion with three parts lipid (usually monoolein [1-oleoyl-rac-glycerol, lot M-239-M8-R]), and the cubic phase forms spontaneously, (2) add precipitant/salt and incubate, and crystals form in hours to weeks. All operations take place at 20 C. 5 The in meso method was proved effective for the purpose of crystal growth.…”

Section: Introductionmentioning

confidence: 99%

“…It constitutes the following two steps for growing crystals: 4,5 (1) mix two parts protein solution/dispersion with three parts lipid (usually monoolein [1-oleoyl-rac-glycerol, lot M-239-M8-R]), and the cubic phase forms spontaneously, (2) add precipitant/salt and incubate, and crystals form in hours to weeks. All operations take place at 20 C. 5 The in meso method was proved effective for the purpose of crystal growth. 1 The method however requires a highly viscous lipid (monoolein) to be mixed with the protein solution (such as bacteriorhodopsin).…”

Section: Introductionmentioning

confidence: 99%

“…The cubic phase is then dispensed into an array of wells on a plate as part of the crystallization setup. 1,5,6 The crystal formed in the wells can be harvested and used to determine the 3D structure of the protein using X-ray crystallography. 7e11 A robotic system for automating the delivery of viscous biosamples into arrays of wells has been built in our laboratory, 10e14 which has significantly increased the efficiency and quality of dispensing.…”

Section: Introductionmentioning

confidence: 99%

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A Microdevice for the Mixing of a Highly Viscous Biosample with Water/Membrane Protein Solution using Microchannel and Centrifugation

Liang

Zheng

2011

JALA: Journal of the Association for Laboratory Automation

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A mechanism for controlling the mixing of highly viscous biosamples at the microliter scale is presented. Existing methods for mixing biosamples using microstirrers or shaking microwells are only effective for non-highly viscous materials. The proposed mechanism mixes monoolein, a highly viscous biosample, with water/membrane protein solution in a microdevice called microcapsule using a microchannel and centrifugation. To achieve effective mixing, the design of the microcapsule along with the microchannel is presented and so is the hydrodynamic model describing the flow of viscous materials in the microchannel. The mixing process is analyzed according to the Reynolds number of the biosamples using computer simulation, which is observed during the experiment using digital images for further analysis. Finally, the new approach is verified by X-ray diffraction experiments with water and the Rh membrane protein solution, which are used to evaluate the effectiveness of mixing. Experimental results not only validate the proposed method but also determine the flow oscillation time in the microchannel to achieve effective and efficient mixing.

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Intrinsic Reactivities of Amino Acids towards Photoalkylation with Benzophenone − A Study Preliminary to Photolabelling of the Transmembrane Protein Glycophorin A

Deseke

Nakatani²,

Ourisson

1998

Eur. J. Org. Chem.

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A systematic study of the photoalkylation of amino acids by benzophenone, a standard photosensitive probe of biomolecules, was performed addressing for the first time chemo‐, regio‐, and stereoselectivities. The high reactivity of the capto‐dative substituted α‐carbon, particularly in glycine, could be demonstrated as well as the chemical lability of the α‐coupling products. Methionine was shown to be favoured both in the sense of reactivity as well as product stability. Preliminary to a project directed towards the elucidation of the topography of glycophorin A in membranes, the present model experiments focussed on the ten amino acids that constitute the transmembrane part of this protein.

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Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Cited by 926 publications

References 40 publications

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

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

A Microdevice for the Mixing of a Highly Viscous Biosample with Water/Membrane Protein Solution using Microchannel and Centrifugation

Intrinsic Reactivities of Amino Acids towards Photoalkylation with Benzophenone − A Study Preliminary to Photolabelling of the Transmembrane Protein Glycophorin A

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