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

Cristallizzazione delle proteine di membrana trasportatrici ABC nelle bicelle lipidiche

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).

Autore in primo piano: Una configurazione di cristallizzazione delle bicelle per le proteine di membrana trasportatrici ABC per far progredire lo sviluppo di farmaci 

Cristallizzazione ABCG5/G8 in ambiente bicellare lipidico per cristallografia a raggi X

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 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  

Trattamento di metilazione riduttiva e pre-cristallizzazione della proteina eterodimerica ABCG5/G8 purificata

Iniziare modificando chimicamente le frazioni proteiche raggruppate attraverso la metilazione riduttiva. Per fare ciò, prendi la proteina purificata e aggiungi 20 millimolari di dimetilammina borano, seguiti da 40 millimolari di formaldeide. Incubare la miscela in agitatori oscillatori per due ore a quattro gradi Celsius.Quindi, aggiungere altri 10 millimolari di dimetilammina borano alla miscela. Successivamente, incubare la miscela per una notte per 12-18 ore a quattro gradi Celsius. Estinguere la reazione aggiungendo 100 millimolari di cloruro tris con pH di 7,5.Utilizzare il tampone di lavaggio e il tampone di eluizione appropriati per caricare la proteina metilata su una colonna di nichel-NTA. Innanzitutto, lavare la colonna di nichel-NTA da due millilitri con 10 volumi di colonna di tampone di lavaggio. E poi, eluire la proteina con il tampone di eluizione.Dopo l'eluizione, far passare l'eluato proteico attraverso una colonna di desalinizzazione PD-10, pre-equilibrata con l'apposito tampone. Aggiungere il colesterolo preparato in isopropanolo o etanolo alla proteina dissalata e incubare la miscela per una notte a quattro gradi Celsius. La mattina successiva, ultracentrifugare la miscela a 150.000 G per 10 minuti a quattro gradi Celsius.Aggiungere il surnatante raccolto a un concentratore centrifugo con un cutoff di 100 kilodalton e concentrare il surnatante a una concentrazione finale di 30-50 milligrammi per millilitro. Ancora una volta, centrifugare la proteina concentrata utilizzando una centrifuga refrigerata da banco ad alta velocità e rimuovere il pellet. Posizionare il surnatante raccolto sul ghiaccio a quattro gradi Celsius prima di procedere all'impostazione della cristallizzazione.Le proteine alchilate conservate a quattro gradi Celsius sono state analizzate mediante cromatografia analitica di filtrazione su gel nel corso di un mese. E ha mostrato una leggera perdita di proteine dopo una settimana.

reductive-methylation-pre-crystallization-treatment-purified

Research

JoVE Journal

Biochemistry

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

314 Views.  University of Ottawa. Iniziare modificando chimicamente le frazioni proteiche raggruppate attraverso la metilazione riduttiva. Per fare ciò, prendi la proteina purificata e aggiungi 20 millimolari di dimetilammina borano, seguiti da 40 millimolari di formaldeide. Incubare la miscela in agitatori oscillatori per due ore a quattro gradi Celsius.Quindi, aggiungere altri 10 millimolari di dimetilammina borano alla miscela. Successivamente, incubare la miscela per una notte per 12-18 ore a quattro gradi Celsius...

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

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 ...

Ricerca

Biochimica

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.

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

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

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.