An efficient nanolitre-volume multi-channel device for highly viscous materials used in membrane protein crystallization

2013 ◽  
Vol 46 (3) ◽  
pp. 829-831
Author(s):  
Jinghui Luo ◽  
Raphaël Zwier ◽  
Jan Pieter Abrahams
Keyword(s):  
Lower Bound ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Crystal Structures ◽  
Protein Crystallization ◽  
Cubic Phase ◽  
Operation Speed ◽  
Membrane Protein Crystallization ◽  
Lipidic Cubic Phase ◽  
Materials Used

The crystal structures of various important membrane proteins could not have been solved without lipidic cubic phase (LCP) crystallization, and yet, compared to traditionalin surfocrystallization, LCP crystallization is not widely used because its extreme viscosity makes the cubic phase difficult to handle. Robots that can dispense LCPs are very specialized and therefore very expensive. Here, an accurate multi-channel device is described. It dispenses LCPs onto glass plates down to volumes of 20 nl accuracy and has an accuracy of 10% when dispensing 200 nl – the lower bound of LCP volumes dispensed for crystallization trials. Because of its multi-channel tips, operation speed goes up by a factor of four compared to simpler devices. It can be operated by hand, but its design also allows it to be built into a basic dispensing robot. Thus, the device lowers the threshold for LCP crystallization of membrane proteins/peptides.

scholarly journals Be Cautious with Crystal Structures of Membrane Proteins or Complexes Prepared in Detergents

Crystals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 86 ◽  
Author(s):  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Crystal Structures ◽  
Structure Determination ◽  
Protein Crystallization ◽  
Energy Coupling ◽  
Careful Examination ◽  
Cubic Phase ◽  
Native Cell ◽  
Membrane Protein Crystallization

Membrane proteins are an important class of macromolecules found in all living organisms and many of them serve as important drug targets. In order to understand their biological and biochemical functions and to exploit them for structure-based drug design, high-resolution and accurate structures of membrane proteins are needed, but are still rarely available, e.g., predominantly from X-ray crystallography, and more recently from single particle cryo-EM — an increasingly powerful tool for membrane protein structure determination. However, while protein-lipid interactions play crucial roles for the structural and functional integrity of membrane proteins, for historical reasons and due to technological limitations, until recently, the primary method for membrane protein crystallization has relied on detergents. Bicelle and lipid cubic phase (LCP) methods have also been used for membrane protein crystallization, but the first step requires detergent extraction of the protein from its native cell membrane. The resulting, crystal structures have been occasionally questioned, but such concerns were generally dismissed as accidents or ignored. However, even a hint of controversy indicates that methodological drawbacks in such structural research may exist. In the absence of caution, structures determined using these methods are often assumed to be correct, which has led to surprising hypotheses for their mechanisms of action. In this communication, several examples of structural studies on membrane proteins or complexes will be discussed: Resistance-Nodulation-Division (RND) family transporters, microbial rhodopsins, Tryptophan-rich Sensory Proteins (TSPO), and Energy-Coupling Factor (ECF) type ABC transporters. These analyses should focus the attention of membrane protein structural biologists on the potential problems in structure determination relying on detergent-based methods. Furthermore, careful examination of membrane proteins in their native cell environments by biochemical and biophysical techniques is warranted, and completely detergent-free systems for membrane protein research are crucially needed.

scholarly journals A fast, simple and robust protocol for growing crystals in the lipidic cubic phase

2012 ◽  
Vol 45 (6) ◽  
pp. 1330-1333 ◽  
Author(s):  
Margaret Aherne ◽  
Joseph A. Lyons ◽  
Martin Caffrey
Keyword(s):  
Quality Control ◽  
Data Collection ◽  
Protein Crystallization ◽  
Cubic Phase ◽  
Sandwich Plates ◽  
Protein Targets ◽  
Test Protein ◽  
Membrane Protein Crystallization ◽  
Lipidic Cubic Phase ◽  
Robust Protocol

A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by thein mesomethod is described. It provides crystals that appear within 15–30 min of setup at 293 K. The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase, which can be daunting as a material when first encountered. Having used the protocol to produce crystals of the test protein, lysozyme, the experimenter can proceed with confidence to apply the method to more valuable membrane (and soluble) protein targets. The glass sandwich plates prepared using this robust protocol can further be used to practice harvesting and snap-cooling ofin meso-grown crystals, to explore diffraction data collection with mesophase-embedded crystals, and for an assortment of quality control and calibration applications when used in combination with a crystallization robot.

scholarly journals Membrane Protein Crystallization In Meso: Lipid Type-Tailoring of the Cubic Phase

2002 ◽  
Vol 83 (6) ◽  
pp. 3393-3407 ◽  
Author(s):  
Vadim Cherezov ◽  
Jeffrey Clogston ◽  
Yohann Misquitta ◽  
Wissam Abdel-Gawad ◽  
Martin Caffrey
Keyword(s):  
Membrane Protein ◽  
Protein Crystallization ◽  
Cubic Phase ◽  
Membrane Protein Crystallization

Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins

2003 ◽  
Vol 36 (6) ◽  
pp. 1372-1377 ◽  
Author(s):  
Vadim Cherezov ◽  
Martin Caffrey
Keyword(s):  
Optical Properties ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Low Cost ◽  
Protein Crystallization ◽  
Batch Crystallization ◽  
Membrane Protein Crystallization ◽  
Crystallization Trial

A simple convenient and low-cost glass-based plate for high-throughput screening of membrane protein crystallization is described. The plates are robust and reduce dramatically the amount of protein and precipitant solution used per crystallization trial, while offering excellent optical properties for the detection of micro-crystals and crystals of colorless proteins. The plates were developed primarily for crystallization of membrane proteins in lipidic mesophases. They can also be used in batch crystallization of soluble and membrane proteins.

A simple and inexpensive nanoliter-volume dispenser for highly viscous materials used in membrane protein crystallization

2005 ◽  
Vol 38 (2) ◽  
pp. 398-400 ◽  
Author(s):  
Vadim Cherezov ◽  
Martin Caffrey
Keyword(s):  
Membrane Proteins ◽  
Protein Crystallization ◽  
Glass Plate ◽  
Screening Process ◽  
Positive Displacement ◽  
Large Numbers ◽  
Membrane Protein Crystallization ◽  
Scale Down ◽  
Materials Used ◽  
Macromolecular Crystallization

Macromolecular crystallization trials involve the testing of large numbers of chemical and environmental conditions. For each, a certain amount of protein and precipitant is required. In the case of membrane proteins, detergent and lipids must be included in the screen, which adds to the number of trials performed. With rare exceptions, membrane proteins come at a premium. Thus, there is a pressing need to scale down the screening process so as to make most efficient use of these scarce materials. Lipids and detergents can also be very expensive and miniaturization is of benefit here too. Recently, a glass-plate system for crystallizing membrane proteins by thein mesomethod was introduced [Cherezov & Caffrey (2003).J. Appl. Cryst.36, 1372–1377; Caffrey (2003).J. Struct. Biol.142, 108–132]. The protocol calls for the handling of small volumes of lipidic/protein dispersions that are extraordinarily viscous. A positive-displacement syringe-based tool was implemented for the preparation and dispensing of these refractory mixtures. However, the device, assembled from commercial parts, operated with microliter-sized samples and was not as economical as it could be in terms of delivered volumes. To effect further miniaturization, and thus conservation of materials, the dispenser has been modified in a way that facilitates accurate and precise repetitive delivery by hand of nanoliter volumes of liquids and of highly viscous dispersions. Here the modifications, which can be made simply and inexpensively to commercially available parts, along with the performance characteristics of the newly configured dispenser, are described

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