scholarly journals Renaturing Membrane Proteins in the Lipid Cubic Phase, a Nanoporous Membrane Mimetic

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Dianfan Li ◽  
Martin Caffrey
Keyword(s):  
Membrane Proteins ◽  
Diacylglycerol Kinase ◽  
Phase Method ◽  
Cubic Phase ◽  
X Ray ◽  
Nanoporous Membrane ◽  
Membrane Mimetic ◽  
Test Protein ◽  
Bicontinuous Cubic

Abstract Membrane proteins play vital roles in the life of the cell and are important therapeutic targets. Producing them in large quantities, pure and fully functional is a major challenge. Many promising projects end when intractable aggregates or precipitates form. Here we show how such unfolded aggregates can be solubilized and the solution mixed with lipid to spontaneously self-assemble a bicontinuous cubic mesophase into the bilayer of which the protein, in a confined, chaperonin-like environment, reconstitutes with 100% efficiency. The test protein, diacylglycerol kinase, reconstituted in the bilayer of the mesophase, was then crystallized in situ by the in meso or lipid cubic phase method providing an X-ray structure to a resolution of 2.55 Å. This highly efficient, inexpensive, simple and rapid approach should find application wherever properly folded, membrane reconstituted and functional proteins are required where the starting material is a denatured aggregate.

scholarly journals In meso in situserial X-ray crystallography of soluble and membrane proteins

2015 ◽  
Vol 71 (6) ◽  
pp. 1238-1256 ◽  
Author(s):  
Chia-Ying Huang ◽  
Vincent Olieric ◽  
Pikyee Ma ◽  
Ezequiel Panepucci ◽  
Kay Diederichs ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
High Throughput ◽  
Protein Structures ◽  
Cubic Phase ◽  
Crystallographic Structure ◽  
Single Digit ◽  
X Ray ◽  
Experimental Phasing ◽  
Glass Plates

The lipid cubic phase (LCP) continues to grow in popularity as a medium in which to generate crystals of membrane (and soluble) proteins for high-resolution X-ray crystallographic structure determination. To date, the PDB includes 227 records attributed to the LCP orin mesomethod. Among the listings are some of the highest profile membrane proteins, including the β2-adrenoreceptor–Gsprotein complex that figured in the award of the 2012 Nobel Prize in Chemistry to Lefkowitz and Kobilka. The most successfulin mesoprotocol to date uses glass sandwich crystallization plates. Despite their many advantages, glass plates are challenging to harvest crystals from. However, performingin situX-ray diffraction measurements with these plates is not practical. Here, an alternative approach is described that provides many of the advantages of glass plates and is compatible with high-throughputin situmeasurements. The novelin meso in situserial crystallography (IMISX) method introduced here has been demonstrated with AlgE and PepT (alginate and peptide transporters, respectively) as model integral membrane proteins and with lysozyme as a test soluble protein. Structures were solved by molecular replacement and by experimental phasing using bromine SAD and native sulfur SAD methods to resolutions ranging from 1.8 to 2.8 Å using single-digit microgram quantities of protein. That sulfur SAD phasing worked is testament to the exceptional quality of the IMISX diffraction data. The IMISX method is compatible with readily available, inexpensive materials and equipment, is simple to implement and is compatible with high-throughputin situserial data collection at macromolecular crystallography synchrotron beamlines worldwide. Because of its simplicity and effectiveness, the IMISX approach is likely to supplant existingin mesocrystallization protocols. It should prove particularly attractive in the area of ligand screening for drug discovery and development.

A Study on the Growth of Cubic GaN Films Using an AlGaAs Buffer Layer Grown on GaAs (100) by Plasma-Assisted Molecular Beam Epitaxy

2000 ◽  
Vol 639 ◽  
Author(s):  
Ryuhei Kimura ◽  
Kiyoshi Takahashi ◽  
H. T. Grahn
Keyword(s):  
Molecular Beam Epitaxy ◽  
Buffer Layer ◽  
Molecular Beam ◽  
High Energy ◽  
Rf Plasma ◽  
Cubic Phase ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cubic Gan

ABSTRACTAn investigation of the growth mechanism for RF-plasma assisted molecular beam epitaxy of cubic GaN films using a nitrided AlGaAs buffer layer was carried out by in-situ reflection high energy electron diffraction (RHEED) and high resolution X-ray diffraction (HRXRD). It was found that hexagonal GaN nuclei grow on (1, 1, 1) facets during nitridation of the AlGaAs buffer layer, but a highly pure, cubic-phase GaN epilayer was grown on the nitrided AlGaAs buffer layer.

scholarly journals Exploring the structural relationship between encapsulated antimicrobial peptides and the bilayer membrane mimetic lipidic cubic phase: studies with gramicidin A′

RSC Advances ◽  
2016 ◽  
Vol 6 (73) ◽  
pp. 68685-68694 ◽  
Author(s):  
Thomas G. Meikle ◽  
Charlotte E. Conn ◽  
Frances Separovic ◽  
Calum J. Drummond
Keyword(s):  
Antimicrobial Peptides ◽  
Low Cost ◽  
Bilayer Membrane ◽  
Structural Relationship ◽  
Gramicidin A ◽  
Cubic Phase ◽  
Membrane Peptides ◽  
Membrane Mimetic ◽  
Lipidic Cubic Phase ◽  
Bicontinuous Cubic

Lipid based bicontinuous cubic mesophases provide a low-cost, robust membrane mimetic nanomaterial which allows for the incorporation of membrane peptides and proteins.

In Situ Synchrotron X-Ray Diffraction Study of the Rhombohedral-to-HT-Cubic Phase Transformation in Ln6 WO12 (Ln = Y, Ho, Er, Yb)

2013 ◽  
Vol 97 (4) ◽  
pp. 1256-1263 ◽  
Author(s):  
Zlatomir D. Apostolov ◽  
Pankaj Sarin ◽  
Robert W. Hughes ◽  
Waltraud M. Kriven
Keyword(s):  
Phase Transformation ◽  
Diffraction Study ◽  
Cubic Phase ◽  
X Ray Diffraction ◽  
X Ray

Small-Angle Neutron and X-ray Scattering from Amphiphilic Stimuli-Responsive Diamond-Type Bicontinuous Cubic Phase

2007 ◽  
Vol 129 (44) ◽  
pp. 13474-13479 ◽  
Author(s):  
Borislav Angelov ◽  
Angelina Angelova ◽  
Vasil M. Garamus ◽  
Geneviève Lebas ◽  
Sylviane Lesieur ◽  
...  
Keyword(s):  
Small Angle ◽  
Cubic Phase ◽  
Stimuli Responsive ◽  
X Ray ◽  
X Ray Scattering ◽  
Bicontinuous Cubic ◽  
Diamond Type ◽  
Ray Scattering

scholarly journals Low-dose in situ prelocation of protein microcrystals by 2D X-ray phase-contrast imaging for serial crystallography

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 1131-1141
Author(s):  
Isabelle Martiel ◽  
Chia-Ying Huang ◽  
Pablo Villanueva-Perez ◽  
Ezequiel Panepucci ◽  
Shibom Basu ◽  
...  
Keyword(s):  
Data Collection ◽  
Phase Contrast ◽  
Low Dose ◽  
Phase Contrast Imaging ◽  
Cubic Phase ◽  
Macromolecular Crystallography ◽  
Contrast Imaging ◽  
X Ray ◽  
Serial Data

Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

Investigating temperature-induced structural changes of lead halide perovskites by in situ X-ray powder diffraction

2019 ◽  
Vol 52 (5) ◽  
pp. 1104-1118
Author(s):  
Rocco Caliandro ◽  
Davide Altamura ◽  
Benny Danilo Belviso ◽  
Aurora Rizzo ◽  
Sofia Masi ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Structural Changes ◽  
Lattice Distortion ◽  
Data Matrix ◽  
Cubic Phase ◽  
X Ray Diffraction ◽  
Temperature Variations ◽  
Temperature Changes ◽  
X Ray

In situ X-ray diffraction experiments offer a unique opportunity to investigate structural dynamics at atomic resolution, by collecting several patterns in an appropriate time sequence (data matrix) while varying the applied stimulus (e.g. temperature changes). Individual measurements can be processed independently by refinement procedures that are based on prior knowledge of the average structure of each crystal phase present in the sample. If the refinement converges, parameters of the average structural model can be assessed and studied as a function of the stimulus variations. An alternative approach consists in applying a multivariate analysis to the data matrix as a whole. Methods such as principal component analysis (PCA) and phase-sensitive detection perform fast, blind and model-independent calculations that can be used for on-site analysis to identify trends in data actually related to the applied stimulus. Both classical and multivariate approaches are here applied to the in situ X-ray diffraction pair distribution function (PDF) setup on two samples of the hybrid perovskite methylammonium (MA) lead iodide obtained by different synthetic routes, subjected to temperature variations. The PDF refinement allows assessing the occurrence of temperature-induced rotations of the PbI6 octahedra and variations in the relative amount of MAPbI3 and intermediate PbI2–MAI–DMSO (dimethyl sulfoxide) crystal phases. A change in the orientation of the methylammonium molecule with temperature is also characterized. Results of the multivariate analysis tools, which include a newly introduced space-dependent variant of PCA, are described, interpreted and validated against simulated data, and their specificity and relation to refinement results are highlighted. The interaction between nearby octahedra is identified as the driving force for the tetragonal-to-cubic phase transition, and three fundamental trends in data having different temperature behaviours are unveiled: (i) irreversible weight-fraction variations of the MAPbI3 and PbI2–MAI–DMSO phases; (ii) reversible structural changes related to the MAPbI3 crystalline phase and its lattice distortion in the ab plane, having the same frequency as the temperature variations; (iii) reversible lattice distortion along the c axis, occurring at twice the frequency of the temperature changes.

In Situ High-Energy X-Ray Diffraction Studies of Melting, Solidification and Solid-State Transformation of Ni3Sn

MRS Advances ◽  
2020 ◽  
Vol 5 (29-30) ◽  
pp. 1529-1535 ◽  
Author(s):  
Rijie Zhao ◽  
Jianrong Gao ◽  
Yang Ren
Keyword(s):  
Solid State ◽  
Elevated Temperatures ◽  
High Energy ◽  
Cubic Phase ◽  
X Ray Diffraction ◽  
State Transformation ◽  
Solid State Transformation ◽  
X Ray ◽  
Melting And Solidification

AbstractMelting, solidification and solid-state transformation of the intermetallic Ni3Sn compound were investigated in situ using synchrotron high-energy X-ray diffraction. It was observed that the compound undergoes a hexagonal to cubic transition before melting. In solidification, a disordered cubic phase crystallizes from the liquid at a large undercooling but it is reordered prior to bulk solidification. In melting and solidification, forced or natural flows are active bringing about significant changes of crystal orientations. These in situ observations provided insights into phase transformations of Ni3Sn at elevated temperatures and their roles in formation of metastable microstructure consisting of coarse grains and subgrains.

scholarly journals In meso in situserial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures

2016 ◽  
Vol 72 (1) ◽  
pp. 93-112 ◽  
Author(s):  
Chia-Ying Huang ◽  
Vincent Olieric ◽  
Pikyee Ma ◽  
Nicole Howe ◽  
Lutz Vogeley ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Data Collection ◽  
Diffraction Data ◽  
Cubic Phase ◽  
Cryogenic Temperatures ◽  
X Ray Crystallography ◽  
Sad Phasing ◽  
Thin Plastic ◽  
G Protein Coupled

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase forin situdiffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase orin mesomethod of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introducedin meso in situserial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.

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