Name: Video: Reductive Methylation and Pre-Crystallization Treatment of Purified Heterodimeric ABCG5/G8 Protein
Uploaded: 2023-08-25T14:40:00
Duration: 2 min 35 s
Description: 339 Views. Reductive Methylation and Pre-Crystallization Treatment of Purified Heterodimeric ABCG5/G8 Protein

reductive methylation and pre-crystallization treatment of purified heterodimeric abcg5&#47;g8 protein

Crystallization of ABC Transporter Membrane Proteins in Lipidic Bicelles

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).

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

Watch this Scientific Journal Video about Reductive Methylation and Pre-Crystallization Treatment of Purified Heterodimeric ABCG5/G8 Protein at JoVE.com

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

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

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Research

JoVE Journal

Biochemistry

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

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

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 ...