Cubic Phase

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Wei Liu - One of the best experts on this subject based on the ideXlab platform.

Vadim Cherezov - One of the best experts on this subject based on the ideXlab platform.

  • preparation of microcrystals in lipidic Cubic Phase for serial femtosecond crystallography
    Nature Protocols, 2014
    Co-Authors: Wei Liu, Andrii Ishchenko, Vadim Cherezov
    Abstract:

    This protocol describes procedures for the preparation and characterization of microcrystals for serial femtosecond crystallography in lipidic Cubic Phase (LCP-SFX) for protein structure determination at X-ray free-electron lasers (XFELs).

  • Membrane protein crystallization in meso: lipid type-tailoring of the Cubic Phase.
    Biophysical journal, 2002
    Co-Authors: Vadim Cherezov, Jeffrey D. Clogston, Y Misquitta, Wissam Abdel-gawad, Martin Caffrey
    Abstract:

    Hydrated monoolein forms the Cubic-Pn3m mesoPhase that has been used for in meso crystallization of membrane proteins. The crystals have subsequently provided high-resolution structures by crystallographic means. It is possible that the hosting Cubic Phase created by monoolein alone, which itself is not a common membrane component, will limit the range of membrane proteins crystallizable by the in meso method. With a view to expanding the range of applicability of the method, we investigated by x-ray diffraction the degree to which the reference Cubic-Pn3m Phase formed by hydrated monoolein could be modified by other lipid types. These included phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, cardiolipin, lyso-PC, a polyethylene glycol-lipid, 2-monoolein, oleamide, and cholesterol. The results show that all nine lipids were accommodated in the Cubic Phase to some extent without altering Phase identity. The positional isomer, 2-monoolein, was tolerated to the highest level. The least well tolerated were the anionic lipids, followed by lyso-PC. The others were accommodated to the extent of 20-25 mol %. Beyond a certain concentration limit, the lipid additives either triggered one or a series of Phase transitions or saturated the Phase and separated out as crystals, as seen with oleamide and cholesterol. The series of Phases observed and their order of appearance were consistent with expectations in terms of interfacial curvature changes. The changes in Phase type and microstructure have been rationalized on the basis of lipid molecular shape, interfacial curvature, and chain packing energy. The data should prove useful in the rational design of Cubic Phase crystallization matrices with different lipid profiles that match the needs of a greater range of membrane proteins.

Martin Caffrey - One of the best experts on this subject based on the ideXlab platform.

  • lipid Cubic Phase as a membrane mimetic for integral membrane protein enzymes
    Proceedings of the National Academy of Sciences of the United States of America, 2011
    Co-Authors: Dianfan Li, Martin Caffrey
    Abstract:

    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.

  • controlling release from the lipidic Cubic Phase amino acids peptides proteins and nucleic acids
    Journal of Controlled Release, 2005
    Co-Authors: Jeffrey D. Clogston, Martin Caffrey
    Abstract:

    Drugs are optimally effective in the therapeutic concentration range. A challenge in the delivery area is to design a system that will allow the therapeutic range to be accessed and to be maintained for defined periods. The lipidic Cubic Phases have been used as delivery matrices with such properties. For water-soluble drugs, release from the Cubic Phase is controlled by transport through aqueous channels that permeate the Phase. Channel size can be tuned over wide limits by adjusting temperature and lipid identity. Thus, the possibility exists to regulate the rate of drug release from the Cubic Phase. With a view to exploiting these features for small molecule, proteinaceous and nucleic acid drugs, we have taken a systematic approach toward understanding how Cubic Phase transport is controlled by Phase identity and microstructure and by the physical and chemical properties of the drug itself. Measurements were made using tryptophan, rubipy, DNA and six proteins as drug surrogates and with three hosting lipids. Remarkably, transport was observed with apo-ferritin whose size far exceeds that of the aqueous channel suggesting a molecular breathing or peristalsis type of facilitated release. Exquisite control over release was achieved by adjusting electrostatic interaction strength and by His-tag displacement.

  • Controlling release from the lipidic Cubic Phase by selective alkylation.
    Journal of controlled release : official journal of the Controlled Release Society, 2005
    Co-Authors: Jeffrey D. Clogston, Gheorghe Craciun, David J. Hart, Martin Caffrey
    Abstract:

    The lipidic Cubic Phase can be viewed as a molecular sponge consisting of interpenetrating nanochannels filled with water and coated by lipid bilayers. It has been used as a delivery matrix for low-molecular-weight drugs. For those that are water-soluble, release is fast and unregulated. This study seeks to exploit the lipid bilayer compartment as a location within the Cubic Phase in which to 'hydrophobically' anchor the water-soluble drug. This was accomplished by controlling partitioning into, and thus release from, the aqueous compartment of the Cubic Phase. Tryptophan was used as a surrogate water-soluble drug and alkylation was implemented to regulate release. By adjusting alkyl chain length, exquisite control was realized. Without alkylation, 20% of the tryptophan was released under standard conditions (infinite sink with a 30-mg Cubic Phase source at pH 7 and 20 degrees C) over a period of 30 min (t(20)). In the case of derivatives with alkyl chains two and eight carbon atoms long, t(20) values of 3 and 13 days, respectively, were observed. Eliminating the charge on tryptophan completely by alkylation produced a derivative that became irreversibly lodged in the lipid bilayer. The release behavior of the short-chain derivatives was mathematically modeled and parameters describing transport have been obtained. Cubic Phase partition coefficients for tryptophan and its derivatives were measured to facilitate modeling. The implications of these findings with regard to the Cubic Phase and related delivery systems, and to vaccine efficacy are discussed.

  • detergents destabilize the Cubic Phase of monoolein implications for membrane protein crystallization
    Biopolymers, 2003
    Co-Authors: Y Misquitta, Martin Caffrey
    Abstract:

    The in meso method for membrane protein crystallization uses a lipidic Cubic Phase as the hosting medium. The Cubic Phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched Cubic Phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the Phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-β-D-glucopyranosides, have on the Phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and Phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40°C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline Phases that might be exploited in low-temperature crystallization. The results show that the Cubic Phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar Phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.

  • Membrane protein crystallization in meso: lipid type-tailoring of the Cubic Phase.
    Biophysical journal, 2002
    Co-Authors: Vadim Cherezov, Jeffrey D. Clogston, Y Misquitta, Wissam Abdel-gawad, Martin Caffrey
    Abstract:

    Hydrated monoolein forms the Cubic-Pn3m mesoPhase that has been used for in meso crystallization of membrane proteins. The crystals have subsequently provided high-resolution structures by crystallographic means. It is possible that the hosting Cubic Phase created by monoolein alone, which itself is not a common membrane component, will limit the range of membrane proteins crystallizable by the in meso method. With a view to expanding the range of applicability of the method, we investigated by x-ray diffraction the degree to which the reference Cubic-Pn3m Phase formed by hydrated monoolein could be modified by other lipid types. These included phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, cardiolipin, lyso-PC, a polyethylene glycol-lipid, 2-monoolein, oleamide, and cholesterol. The results show that all nine lipids were accommodated in the Cubic Phase to some extent without altering Phase identity. The positional isomer, 2-monoolein, was tolerated to the highest level. The least well tolerated were the anionic lipids, followed by lyso-PC. The others were accommodated to the extent of 20-25 mol %. Beyond a certain concentration limit, the lipid additives either triggered one or a series of Phase transitions or saturated the Phase and separated out as crystals, as seen with oleamide and cholesterol. The series of Phases observed and their order of appearance were consistent with expectations in terms of interfacial curvature changes. The changes in Phase type and microstructure have been rationalized on the basis of lipid molecular shape, interfacial curvature, and chain packing energy. The data should prove useful in the rational design of Cubic Phase crystallization matrices with different lipid profiles that match the needs of a greater range of membrane proteins.

Petra Fromme - One of the best experts on this subject based on the ideXlab platform.

Uwe Weierstall - One of the best experts on this subject based on the ideXlab platform.

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