Dataset Information

X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization.

ABSTRACT: Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5?Å.

SUBMITTER: Schieferstein JM

PROVIDER: S-EPMC5403737 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Publications

X-ray transparent microfluidic chips for high-throughput screening and optimization of <i>in meso</i> membrane protein crystallization.

Schieferstein Jeremy M JM Pawate Ashtamurthy S AS Sun Chang C Wan Frank F Sheraden Paige N PN Broecker Jana J Ernst Oliver P OP Gennis Robert B RB Kenis Paul J A PJA

Biomicrofluidics 20170301 2

Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as <i>in meso</i> crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automa ...[more]

PMID: 28469762

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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

X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization.

Publications

X-ray transparent microfluidic chips for high-throughput screening and optimization of <i>in meso</i> membrane protein crystallization.

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