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Microfluidic Generation of Lipidic Mesophases for Membrane Protein Crystallization.

ABSTRACT: We report on a microfluidic method for the formation of aqueous/lipid mesophases to enable screening of suitable crystallization conditions of membrane proteins from a membrane-like phase in sub-20 nanoliter volumes. This integrated microfluidic chip and the employed mixing strategy address the specific challenges associated with the mixing of fluids of highly different viscosities (here a factor of 30) as well as the non-Newtonian character of the resulting mesophases. The chip requires less than 20 nL of material per condition screened whereas typically on the order of 10 ?L or more is needed for a batch preparation in the present screening methods. We validated our approach with the successful crystallization of the membrane protein bacteriorhodopsin.

SUBMITTER: Perry SL

PROVIDER: S-EPMC2721472 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Microfluidic Generation of Lipidic Mesophases for Membrane Protein Crystallization.

Perry Sarah L SL Roberts Griffin W GW Tice Joshua D JD Gennis Robert B RB Kenis Paul J A PJ

Crystal growth & design 20090601 6

We report on a microfluidic method for the formation of aqueous/lipid mesophases to enable screening of suitable crystallization conditions of membrane proteins from a membrane-like phase in sub-20 nanoliter volumes. This integrated microfluidic chip and the employed mixing strategy address the specific challenges associated with the mixing of fluids of highly different viscosities (here a factor of 30) as well as the non-Newtonian character of the resulting mesophases. The chip requires less th ...[more]

PMID: 20161169

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Project description:The crystal structure of the β(2)-adrenergic receptor in complex with an agonist and its cognate G protein has just recently been determined. It is now possible to explore in molecular detail the means by which this paradigmatic transmembrane receptor binds agonist, communicates the impulse or signaling event across the membrane, and sets in motion a series of G protein-directed intracellular responses. The structure was determined using crystals of the ternary complex grown in a rationally designed lipidic mesophase by the so-called in meso method. The method is proving to be particularly useful in the G protein-coupled receptor field where the structures of 13 distinct receptor types have been determined in the past 5 years. In addition to receptors, the method has proven to be useful with a wide variety of integral membrane protein classes that include bacterial and eukaryotic rhodopsins, light-harvesting complex II (LHII), photosynthetic reaction centers, cytochrome oxidases, β-barrels, an exchanger, and an integral membrane peptide. This attests to the versatility and range of the method and supports the view that the in meso method should be included in the arsenal of the serious membrane structural biologist. For this to happen, however, the reluctance to adopt it attributable, in part, to the anticipated difficulties associated with handling the sticky, viscous cubic mesophase in which crystals grow must be overcome. Harvesting and collecting diffraction data with the mesophase-grown crystals are also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. Over the years, we have endeavored to establish how the method works at a molecular level and to make it user-friendly. To these ends, tools for handling the mesophase in the pico- to nanoliter volume range have been developed for highly efficient crystallization screening in manual and robotic modes. Methods have been implemented for evaluating the functional activity of membrane proteins reconstituted into the bilayer of the cubic phase as a prelude to crystallogenesis. Glass crystallization plates that provide unparalleled optical quality and sensitivity to nascent crystals have been built. Lipid and precipitant screens have been designed for a more rational approach to crystallogenesis such that the method can now be applied to an even wider variety of membrane protein types. In this work, these assorted advances are outlined along with a summary of the membrane proteins that have yielded to the method. The prospects for and the challenges that must be overcome to further develop the method are described.

Project description:Lipidic mesophases are a class of highly ordered soft materials that form when certain lipids are mixed with water. Understanding the relationship between the composition and the microstructure of mesophases is necessary for fundamental studies of self-assembly in amphiphilic systems and for applications, such as the crystallization of membrane proteins. However, the laborious formulation protocol for highly viscous mesophases and the large amounts of material required for sample formulation are significant obstacles in such studies. Here we report a microfluidic platform that facilitates investigations of the phase behavior of mesophases by reducing sample consumption 300-fold, and automating and parallelizing sample formulation. The mesophases were formulated on-chip using less than 80 nL of material per sample and their microstructure was analyzed in situ using small-angle X-ray scattering (SAXS). The 220 ?m-thick X-ray compatible platform was comprised of thin polydimethylsiloxane (PDMS) layers sandwiched between cyclic olefin copolymer (COC) sheets. Uniform mesophases were prepared using an active on-chip mixing strategy coupled with periodic cooling of the sample to reduce viscosity. We validated the platform by preparing and analyzing mesophases of the lipid monoolein (MO) mixed with aqueous solutions of different concentrations of ?-octylglucoside (?OG), a detergent frequently used in membrane protein crystallization. Four samples were prepared in parallel on chip, by first metering and automatically diluting ?OG to obtain detergent solutions of different concentration, then metering MO, and finally mixing by actuation of pneumatic valves. Integration of detergent dilution and subsequent mixing significantly reduced the number of manual steps needed for sample preparation. Three different types of mesophases typical for MO were successfully identified in SAXS data from on-chip samples. Microstructural parameters of identical samples formulated in different chips showed excellent agreement. Phase behavior of samples on-chip (~80 nL per sample) corresponded well with that of samples prepared via the traditional coupled-syringe method using at least two orders of magnitude more material ("off-chip", 35-40 ?L per sample), further validating the applicability of the microfluidic platform for on-chip characterization of mesophase microstructure.

Dataset Information

Microfluidic Generation of Lipidic Mesophases for Membrane Protein Crystallization.

Publications

Microfluidic Generation of Lipidic Mesophases for Membrane Protein Crystallization.

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