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Current methods in structural proteomics and its applications in biological sciences

View Article: PubMed Central

ABSTRACT

A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation.

No MeSH data available.


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Protein crystallization and automation. a TTP LabTech’s mosquito® Crystal automates protein crystallography vapor diffusion set-ups, additive screening and microseeding; b TTP LabTech’s mosquito® LCP: a dedicated instrument for crystallising membrane proteins using lipidic cubic phase screening. The panel highlights the positive displacement syringe, which dispenses the highly viscous lipid mesophases used in the LCP technique into 96-well crystallization plates. (c and d) Crystallization plate set up for hanging drop vapor diffusion experiments; e nanoliter sitting drop experiments set up in a 96-well plate. (Images courtesy of TTP LabTech Ltd, UK)
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Fig4: Protein crystallization and automation. a TTP LabTech’s mosquito® Crystal automates protein crystallography vapor diffusion set-ups, additive screening and microseeding; b TTP LabTech’s mosquito® LCP: a dedicated instrument for crystallising membrane proteins using lipidic cubic phase screening. The panel highlights the positive displacement syringe, which dispenses the highly viscous lipid mesophases used in the LCP technique into 96-well crystallization plates. (c and d) Crystallization plate set up for hanging drop vapor diffusion experiments; e nanoliter sitting drop experiments set up in a 96-well plate. (Images courtesy of TTP LabTech Ltd, UK)

Mentions: Methods used for crystallization include vapor diffusion, batch crystallization, dialysis, seeding, free-interface diffusion and temperature-induced crystallization. The most popular method for setting up crystallization experiments is vapor diffusion, which includes hanging drop (for smaller volumes), sitting drop (for larger volumes), the sandwich drop, reverse vapor diffusion and pH gradient vapor diffusion methods. A drop containing a mixture of precipitant and protein solution is sealed in a chamber with pure precipitant. Water vapor subsequently diffuses from the drop until the osmolarity of the drop and the precipitant is equal. The dehydration of the drop causes a slow concentration change of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram (Dessau and Modis 2011). Batch crystallization relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant. The batch method is usually carried out under oil to prevent the diffusion of water out of the drop (Chayen 1997). Many of these methods can be performed using HT automated instrumentation and miniaturization of crystallization experiments and have had huge impacts on protein crystallization in terms of saving time and conserving precious sample. For example, crystallization robots such as the Phoenix™ RE (Rigaku Corporation) and the Mosquito® (TTP Labtech), which can accurately and reproducibly dispense very small volumes (nl in size) into 96-well plates for automated screening and optimization of crystallization conditions, are now commonplace in many laboratories (Fig. 4). In addition, TTP LabTech’s Mosquito®LCP (Lipid Cubic Phase) has been designed to aid in the crystallization of membrane proteins by accurately dispensing nanoliter quantities of highly viscous lipids or detergents that are required to retain the structural integrity of the sample. A recent development in protein crystallization has been the use of high-density, chip-based microfluidic systems for crystallizing proteins using the free-interface diffusion method at nanoliter scale, including Emerald Biosystems MPCS (Microcapillary Protein Crystallization System)(Gerdts et al. 2008), Fluidigm Corporations TOPAZ® system (Segelke 2005) and the Microlytic Crystal Former (Stojanoff et al. 2011). These platforms have the advantage of using minimal protein sample to screen a broad range of crystallization conditions. The Rigaku CrystalMation™ system was set up to fully automate the crystallization process while dealing with sample volumes of 100 nl per experiment.Fig. 4


Current methods in structural proteomics and its applications in biological sciences
Protein crystallization and automation. a TTP LabTech’s mosquito® Crystal automates protein crystallography vapor diffusion set-ups, additive screening and microseeding; b TTP LabTech’s mosquito® LCP: a dedicated instrument for crystallising membrane proteins using lipidic cubic phase screening. The panel highlights the positive displacement syringe, which dispenses the highly viscous lipid mesophases used in the LCP technique into 96-well crystallization plates. (c and d) Crystallization plate set up for hanging drop vapor diffusion experiments; e nanoliter sitting drop experiments set up in a 96-well plate. (Images courtesy of TTP LabTech Ltd, UK)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376864&req=5

Fig4: Protein crystallization and automation. a TTP LabTech’s mosquito® Crystal automates protein crystallography vapor diffusion set-ups, additive screening and microseeding; b TTP LabTech’s mosquito® LCP: a dedicated instrument for crystallising membrane proteins using lipidic cubic phase screening. The panel highlights the positive displacement syringe, which dispenses the highly viscous lipid mesophases used in the LCP technique into 96-well crystallization plates. (c and d) Crystallization plate set up for hanging drop vapor diffusion experiments; e nanoliter sitting drop experiments set up in a 96-well plate. (Images courtesy of TTP LabTech Ltd, UK)
Mentions: Methods used for crystallization include vapor diffusion, batch crystallization, dialysis, seeding, free-interface diffusion and temperature-induced crystallization. The most popular method for setting up crystallization experiments is vapor diffusion, which includes hanging drop (for smaller volumes), sitting drop (for larger volumes), the sandwich drop, reverse vapor diffusion and pH gradient vapor diffusion methods. A drop containing a mixture of precipitant and protein solution is sealed in a chamber with pure precipitant. Water vapor subsequently diffuses from the drop until the osmolarity of the drop and the precipitant is equal. The dehydration of the drop causes a slow concentration change of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram (Dessau and Modis 2011). Batch crystallization relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant. The batch method is usually carried out under oil to prevent the diffusion of water out of the drop (Chayen 1997). Many of these methods can be performed using HT automated instrumentation and miniaturization of crystallization experiments and have had huge impacts on protein crystallization in terms of saving time and conserving precious sample. For example, crystallization robots such as the Phoenix™ RE (Rigaku Corporation) and the Mosquito® (TTP Labtech), which can accurately and reproducibly dispense very small volumes (nl in size) into 96-well plates for automated screening and optimization of crystallization conditions, are now commonplace in many laboratories (Fig. 4). In addition, TTP LabTech’s Mosquito®LCP (Lipid Cubic Phase) has been designed to aid in the crystallization of membrane proteins by accurately dispensing nanoliter quantities of highly viscous lipids or detergents that are required to retain the structural integrity of the sample. A recent development in protein crystallization has been the use of high-density, chip-based microfluidic systems for crystallizing proteins using the free-interface diffusion method at nanoliter scale, including Emerald Biosystems MPCS (Microcapillary Protein Crystallization System)(Gerdts et al. 2008), Fluidigm Corporations TOPAZ® system (Segelke 2005) and the Microlytic Crystal Former (Stojanoff et al. 2011). These platforms have the advantage of using minimal protein sample to screen a broad range of crystallization conditions. The Rigaku CrystalMation™ system was set up to fully automate the crystallization process while dealing with sample volumes of 100 nl per experiment.Fig. 4

View Article: PubMed Central

ABSTRACT

A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation.

No MeSH data available.


Related in: MedlinePlus