Lipídic Bicelle विधि का प्रयोग झिल्ली प्रोटीन की उच्च throughput Crystallization
Summary
Bicelles लिपिड / amphiphile मिश्रण है कि एक लिपिड bilayer के भीतर झिल्ली प्रोटीन (सांसदों) को बनाए रखने, लेकिन अद्वितीय चरण व्यवहार है कि crystallization रोबोटों द्वारा उच्च throughput स्क्रीनिंग की सुविधा है. इस तकनीक को सफलतापूर्वक दोनों prokaryotic और यूकेरियोटिक स्रोतों से उच्च संकल्प संरचनाओं के एक नंबर का उत्पादन किया. इस वीडियो lipídic bicelle मिश्रण पैदा bicelle मिश्रण में सांसदों को शामिल माध्यम से crystallizations (मैन्युअल रूप में अच्छी तरह के रूप में robotically) परीक्षण और कटाई क्रिस्टल स्थापित करने के लिए प्रोटोकॉल का वर्णन करता है.
Abstract
Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity.
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent′s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots.
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer′s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.
Protocol
Discussion
Bicelles एक अद्वितीय lipídic मीडिया है कि एक देशी bilayer की तरह पर्यावरण की पेशकश करते हुए बर्ताव के रूप में यदि डिटर्जेंट द्वारा solubilized हैं. इस संपत्ति bicelles अन्य लिपिड आधारित crystallization तरीकों पर एक विशिष्ट लाभ देता है के बा…
Divulgations
The authors have nothing to disclose.
Acknowledgements
हम डीआरएस धन्यवाद देना चाहूंगा. जेम्स बॉवी और bicelle विधि और डा. अवीव पाज़ पर उपयोगी विचार – विमर्श के लिए तकनीकी विशेषज्ञता और मार्गदर्शन प्रदान करने के लिए सलेम Faham. हम समर्थन के लिए प्रयोगात्मक ले दू स्वीकार करते हैं. रचना Ujwal MemX बायोसाइंसेज LLC, जो, हालांकि, इस काम का समर्थन नहीं किया में वित्तीय हित है. इस काम के हिस्से में एनआईएच (GM078844 RO1) से अनुदान द्वारा समर्थित किया गया था.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| DMPC | Affymetrix | D514 | |
| CHAPSO | Affymetrix | C317 | |
| Ready-to-use Bicelles | MemX Biosciences | MX201001/MX201002 | |
| Crystallization Screens | Qiagen, Hamptop Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience | Standard commercially available screens can be used for initial screening | |
| Crystallization Set-up | Standard manual and/or robotic set-up available in lab can be used. |
References
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