Bicelles هي مزيج الدهن / مزدوج الألفة التي تحافظ على بروتينات غشاء (النواب) في طبقة ثنائية المادة الدهنية ولكن فريد السلوك المرحلة التي تسهل سرعة عالية الفحص بواسطة الروبوتات التبلور. وقد أنتجت هذه التقنية بنجاح في عدد من هياكل عالية الدقة من مصادر حقيقية النواة وبدائية. هذا الفيديو يصف بروتوكولات لتوليد الخليط bicelle lipidic ، وإدماج النواب في خليط bicelle ، وإقامة المحاكمات التبلور (يدويا وكذلك آليا) ، والحصاد بلورات من المتوسط.
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.
Bicelles هي فريدة من نوعها وسائل الإعلام التي تقدم lipidic الأصلي طبقة ثنائية مثل البيئة في حين تتصرف كما لو solubilized بواسطة المنظفات. هذه الخاصية يعطي bicelles ميزة واضحة اكثر من غيرها من أساليب بلورة الدهون على أساس أنه ليس هناك أي منحنى التعلم أو المعدات المتخصصة اللازمة لهذه ا?…
The authors have nothing to disclose.
نود أن نشكر الدكاترة. جيمس باوي والفحام سالم لتقديم الخبرة الفنية والمشورة بشأن طريقة bicelle تل باز ، والدكتور لإجراء مناقشات مفيدة. نعترف لو دو لدعم التجريبية. راتشنا Ujwal ومصلحة مالية في MemX العلوم البيولوجية المحدودة ، التي ، مع ذلك ، لم تؤيد هذا العمل. وأيد هذا العمل في جزء من المنح المقدمة من المعاهد الوطنية للصحة (RO1 GM078844).
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. |