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Effects of detergent ?-octylglucoside and phosphate salt solutions on phase behavior of monoolein mesophases.

ABSTRACT: Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent ?-octylglucoside (?OG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with ?OG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (L?, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods.

SUBMITTER: Khvostichenko DS

PROVIDER: S-EPMC3797584 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Effects of detergent β-octylglucoside and phosphate salt solutions on phase behavior of monoolein mesophases.

Khvostichenko Daria S DS Ng Johnathan J D JJ Perry Sarah L SL Menon Monisha M Kenis Paul J A PJ

Biophysical journal 20131001 8

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent β-octylglucoside (βOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures o ...[more]

PMID: 24138861

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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.

Project description:Octacalcium phosphate (OCP), a new-generation bone substitute material, is a considered precursor of the biological bone apatite. The two-layered structure of OCP contains the apatitic and hydrated layers and is intensively involved in ion-exchange surface reactions, which results in OCP hydrolysis to hydroxyapatite and adsorption of ions or molecular groups presented in the environment. During various in vitro procedures, such as biomaterial solubility, additive release studies, or the functionalization technique, several model solutions are applied. The composition of the environmental solution affects the degree and rate of OCP hydrolysis, its surface reactivity, and further in vitro and in vivo properties. The performed study was aimed to track the structural changes of OCP-based materials while treating in the most popular model solutions of pH values 7.2-7.4: simulated body fluid (SBF), Dulbecco's phosphate-buffered saline (DPBS), supersaturated calcification solution (SCS), normal saline (NS), and Dulbecco's modified Eagle's medium (DMEM). Various degrees of OCP hydrolysis and/or precipitate formation were achieved through soaking initial OCP granules in the model solutions. Detailed data of X-ray diffraction, Fourier-transform infrared spectroscopy, atomic emission spectrometry with inductively coupled plasma, and scanning electron microscopy are presented. Cultivation of osteosarcoma cells was implemented on OCP pre-treated in DMEM for 1-28 days. It was shown that NS mostly degraded the OCP structure. DPBS slightly changed the OCP structure during the first treatment term, and during further terms, the crystals got thinner and OCP hydrolysis took place. Treatment in SBF and SCS caused the precipitate formation along with OCP hydrolysis, with a larger contribution of SCS solution to precipitation. Pre-treating in DMEM enhanced the cytocompatibility of materials. As a result, on performing the in vitro procedures, careful selection of the contact solution should be made to avoid the changes in materials structure and properties and get adequate results.

Dataset Information

Effects of detergent ?-octylglucoside and phosphate salt solutions on phase behavior of monoolein mesophases.

Publications

Effects of detergent β-octylglucoside and phosphate salt solutions on phase behavior of monoolein mesophases.

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