crystallization of heterodimeric abcg5&#47;g8 protein in lipidic bicelle using hanging drop vapor diffusion

脂质双细胞中 ABC 转运蛋白膜蛋白的结晶

ATP-binding cassette (ABC) transporters constitute a superfamily of membrane proteins responsible for diverse ATP-dependent transport processes across biological membranes1,2,3,4,5. These transporter proteins are implicated in cardiovascular diseases and play a significant role in facilitating cholesterol efflux to the bile for subsequent excretion in the liver. Consequently, cholesterol metabolism and balance have garnered considerable interest over the years6. A specific mechanism involved in the elimination of cholesterol and other sterols from the body involves members of the human ABCG subfamily, notably the heterodimeric ABCG5/G87,8,9,10. Mutations in either of these genes disrupt the heterodimer, leading to loss of function and causing sitosterolemia, a disorder affecting sterol trafficking11,12,13. Given the disease&#39;s relevance and their role in promoting cholesterol efflux, sterol transporters have attracted significant attention. Nevertheless, the intricate details of their molecular mechanism and substrate selectivity remain largely undisclosed. Thus, the elucidation of the crystal structure of ABCG5/G8 is a crucial stride toward comprehending the mechanisms and downstream functions in cholesterol transport.
Membrane proteins require anchoring within membranes to fold and function correctly. Consequently, extracting membrane proteins from their natural environment often results in protein instability, misfolding, and loss of function14,15. These challenges underscore the primary hurdles faced in membrane protein crystallization. However, the reconstitution of proteins into synthetic detergent bilayers, like bicelles, has emerged as a solution to this predicament, enabling the maintenance of membrane proteins within a native-like bilayer milieu16. Bicelles are assemblies of synthetic phospholipids and detergents suspended and solubilized in water. Notably, they adopt a bilayer structure that mimics biological membranes16,17,18. Bicelles can transition between liquid and gel phases based on temperature and viscosity. Bicelle crystallization capitalizes on the small bilayer discs and low viscosity at reduced temperatures, facilitating thorough mixing of proteins and bicelle solutions. The size of the bicelles depends on the detergent-to-lipid ratio during preparation19,20. The prevalent detergents for bicelle formation include 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), along with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 1,2-ditridecanoyl-sn-glycerol-3-phosphocholine (DHPC)21. These detergents are used in conjunction with lipids such as di-myristoyl-phosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Furthermore, recent studies have demonstrated the full functionality of membrane proteins within bicelles under physiological conditions. For example, Lee and colleagues successfully crystallized and reported the crystal structure of ABCG5/ABCG8 based on a lipid bilayer22,23. In the crystallization process, protein-bicelle mixtures can be accommodated using standard equipment, including high-throughput crystallization robots24. The feasibility of utilizing bicelles, however, hinges on the proteins&#39; thermostability due to the crystallization conditions at higher temperatures. Nevertheless, when compared to other techniques, the requisite crystallization conditions for membrane proteins generally remain mild, involving low concentrations of precipitant, salt, and buffer. This renders both protein-bicelle mixtures and vapor diffusion effective and easily implementable tools for structural studies of membrane proteins.
This protocol outlines essential steps in protein preparation and bicelle crystallization for determining the X-ray crystal structure of ABCG5/G8 at high resolution (Figure 1).

作者聚焦：用于 ABC 转运蛋白转运蛋白的 Bicelle 结晶设置以推进药物开发 

用于 X 射线晶体学的脂质双铈环境中的 ABCG5/G8 结晶

Hello, my team study type of protein loss span across the cellular membrane, and we are looking at the relationship to the medicine and biology. Specifically, we are looking at one particular protein which would require ATP to drive its function, and then it, the name is called ATP binding cassette transporter. In particular, we are looking at their fundamental structure function relationship, and then use that to understand a lot of chronic disease, especially cardiovascular disease.Due to the complexity of purifying the membrane proteins and their stability at native temperatures, obtaining 3D protein crystals is the main challenge in the field, and it also limits the drug discovery applications. Our team has established methods in cryo-EM and X-Ray crystallography for the structural studies of membrane transporter proteins. And previously our lab also solved the crystal structure of the first human ABCG5/G8 obligate heterodimer, in both apo as well as cholesterol-bound states.Our results can provide a breakthrough in small molecule drug discovery or drug repurposing. Obtaining 3D protein crystals can also enable soaking experiments for the cocrystallization of protein and ligand complexes. Our laboratory aims to determine the molecular basis of lipid recognition and translocation by ATP dependent lipid transporter proteins.And we also aim to establish new methods in drug development for studying the molecular dynamics and structural aspects of ABC transporters.

Watch this Scientific Journal Video about Crystallization of Heterodimeric ABCG5/G8 Protein in Lipidic Bicelle Using Hanging Drop Vapor Diffusion at JoVE.com

Crystallization of Heterodimeric ABCG5/G8 Protein in Lipidic Bicelle Using Hanging Drop Vapor Diffusion  

使用悬滴蒸气扩散法结晶脂质二聚体ABCG5/G8蛋白

首先制备含有脂质和洗涤剂的 10% bicelle 储备溶液。为此，取预先干燥的脂质，即 5 摩尔百分比胆固醇和 95 摩尔百分比 DMPC 的混合物，并在其中加入去离子水中的 CHAPSO 洗涤剂溶液。使用水浴超声仪，通过在连续通电下在冰冷的水中超声处理脂质洗涤剂混合物，使其重悬，直到溶液澄清。然后借助 0.2 微米离心过滤器，去除任何未溶解的成分。接下来，在冰上工作，将所得的bicelle储备溶液与预先制备的蛋白质样品以1比4的体积比混合。将蛋白质二烯混合物在冰上孵育 30 分钟。对于结晶，在48孔板中将0.5或1微升的蛋白质二烯混合物与等体积的结晶储液库溶液混合。然后使用 48 孔板在悬挂式液滴蒸气扩散模式下设置结晶，并在 20 摄氏度下孵育结晶装置。第二天，检查结晶托盘，确保盖玻片密封正确。每天至少使用配备偏光片的台式体视显微镜检查晶体生长一次。最后，将蛋白质晶体浸泡在 0.2 摩尔苹果酸钠中，并使用液氮将它们快速冷冻在 50 或 100 微米的冷冻环中。适合数据收集的成熟晶体通常在一到两周内出现，尺寸通常为 50 微米 x 100 微米 x 2 微米。

crystallization-heterodimeric-abcg5g8-protein-lipidic-bicelle-using

Research

JoVE Journal

Biochemistry

Crystallization of Heterodimeric ABCG5&#47;G8 Protein in Lipidic Bicelle Using Hanging Drop Vapor Diffusion

303 次观看.  University of Ottawa. 首先制备含有脂质和洗涤剂的 10% bicelle 储备溶液。为此，取预先干燥的脂质，即 5 摩尔百分比胆固醇和 95 摩尔百分比 DMPC 的混合物，并在其中加入去离子水中的 CHAPSO 洗涤剂溶液。使用水浴超声仪，通过在连续通电下在冰冷的水中超声处理脂质洗涤剂混合物，使其重悬，直到溶液澄清。然后借助 0.2 微米离心过滤器，去除任何未溶解的成分。接下来，在冰上工作，将所?...

视频: Crystallization of Heterodimeric ABCG5/G8 Protein in Lipidic Bicelle Using Hanging Drop Vapor Diffusion  

ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

This protocol describes a setup for the crystallization of the sterol transporter ABCG5/G8. ABCG5/G8 is reconstituted into bicelles for hanging-drop crystallization. The protocol does not require specialized materials or substrates, making it accessible and easy to adapt in any laboratory for determining the protein structure through X-ray crystallography.

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an ...

科研

生物化学

Reductive Methylation and Pre-Crystallization Treatment of Purified Heterodimeric ABCG5&#47;G8 Protein

Reductive Methylation and Pre-Crystallization Treatment of Purified Heterodimeric ABCG5/G8 Protein  

Begin by chemically modifying the pooled protein fractions through reductive methylation. To do so, take the purified protein and add 20 millimolar dimethylamine borane, followed by 40 millimolar formaldehyde to it. Incubate the mixture in oscillatory shaker for two hours at four degrees celsius.Then, add an additional 10 millimolar dimethylamine borane to the mixture. Next, incubate the mixture overnight for 12 to 18 hours at four degrees Celsius. Quench the reaction by adding 100 millimolar tris chloride having a pH of 7.5.Use the appropriate wash buffer and elution buffer for loading the methylated protein onto a nickel-NTA column. First, wash the two milliliter nickel-NTA column with 10 column volumes of wash buffer. And then, elute the protein with the elution buffer.Following elution, pass the protein eluate through a PD-10 desalting column, pre-equilibrated with the appropriate buffer. Add cholesterol prepared in isopropanol or ethanol to the desalted protein, and incubate the mixture overnight at four degrees Celsius. The next morning, ultracentrifuge the mixture at 150, 000G for 10 minutes at four degrees Celsius.Add the collected supernatant to a centrifugal concentrator with a 100 kilodalton cutoff, and concentrate the supernatant to a final concentration of 30 to 50 milligrams per milliliter. Once more, centrifuge the concentrated protein using a high speed benchtop refrigerated centrifuge, and remove the pellet. Place the collected supernatant on the ice at four degrees Celsius before proceeding to set the crystallization.Alkylated proteins stored at four degrees Celsius were analyzed by analytical gel filtration chromatography over the course of a month. And it showed a slight protein loss after a week.

纯化异二聚体ABCG5/G8蛋白的还原甲基化和预结晶处理

Begin by preparing a 10%bicelle stock solution containing the lipids and detergent. To do so, take the pre-dried lipid, which is a mix of five mole percent cholesterol and 95 mole percent DMPC, and add the CHAPSO detergent solution in deionized water to it. Using a water bath sonicater, resuspend the lipid detergent mixture by sonicating it in ice-chilled water under continuous power until the solution is clear.Then with the aid of a 0.2 micron centrifugal filter, remove any undissolved components. Next, working on the ice, combine the resultant bicelle stock solution with the pre-prepared protein sample at a one to four volumetric ratio. Incubate the protein bicelle mixture on ice for 30 minutes.For crystallization, in a 48 well plate mix 0.5 or one microliter of the protein bicelle mixture with an equal volume of crystallization reservoir solution. Then set up crystallization in a hanging drop vapor diffusion mode using the 48 well plate and incubate the crystallization setup at 20 degrees Celsius. On the following day, check the crystallization trays to ensure the cover glass is sealed properly.Check on the crystal growth at least once daily using a tabletop stereo microscope equipped with a polarizer. Lastly, soak the protein crystals in 0.2 molar sodium malate and flash freeze them in 50 or 100 micrometer cryo loops using liquid nitrogen. Mature crystals that were suitable for data collection usually appeared within one to two weeks and generally had the dimensions of 50 microns by 100 microns by two microns.