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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.


Examples for widely used structure determination methods. a Structure of E. coli Arabinose Isomerase (PDB 2AJT) determined by single-wavelength anomalous diffraction (SAD). Selenomithionine residues are also shown (Manjasetty and Chance 2006). b Structure of DAPK3 (PDB 2J90) determined with the molecular replacement (MR) method using the template prepared by homolog structures (PDB 1YRT, 1JKT, 1WVX) (Pike et al. 2008)
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Fig7: Examples for widely used structure determination methods. a Structure of E. coli Arabinose Isomerase (PDB 2AJT) determined by single-wavelength anomalous diffraction (SAD). Selenomithionine residues are also shown (Manjasetty and Chance 2006). b Structure of DAPK3 (PDB 2J90) determined with the molecular replacement (MR) method using the template prepared by homolog structures (PDB 1YRT, 1JKT, 1WVX) (Pike et al. 2008)

Mentions: Heavy-atom incorporation (isomorphous replacement, anomalous scattering and anomalous dispersion), molecular replacement and direct methods are commonly used techniques to solve protein structures. The general requirement for the exploitation of the anomalous signal for the determination of phase estimations via multiple or single-wavelength anomalous diffraction (MAD or SAD) techniques is that the protein crystal should contain anomalously scattering atoms, e.g., Hg, Pt or Se. With the advent of tunable X-ray sources and improved data collection techniques, it is now possible to measure the intensities of diffracted X-rays with very high precision. The small differences in intensities between Bijovoet pairs due to the presence of heavy atoms can be used to calculate initial estimates of the protein phase angle. One of the strategies widely used for the determination of novel protein structures is selenomethionine incorporation, where selenomethionine is replaced by methionine in the protein during expression. This method has revolutionized protein X-ray crystallography and it is estimated that over two-thirds of all novel crystal structures have been determined using either Se-SAD or Se-MAD (Fig. 7a). Novel structures can also be solved using the weak anomalous signals from atoms, such as sulfur and phosphorous present in certain macromolecules. SAD represents the most commonly used technique for novel proteins in SP centers. Multiple or single isomorphous replacement (MIR or SIR) methods also require the introduction of heavy atoms such as mercury, platinum, uranium or gold into the macromolecule under investigation. These heavy atoms must be incorporated into protein crystals without disrupting the lattice interactions so that it remains isomorphous with respect to the native crystal. In the SIR method, intensity differences between the heavy-atom derivatized and native crystal are used to calculate experimental phases. Recently, the SIR phasing protocol has been re-applied in the radiation damage-induced phasing (RIP) technique, where the differences in intensities induced by radiation damage are used as a phasing tool (Ravelli et al. 2003). Limitations of these phasing protocols are mainly due to the deleterious effect that a high X-ray dose has on a protein crystal. X-ray radiation damage induces many changes to the protein structure and to the solvent, resulting in a consistent number of damaged sites and a decrease in the diffraction quality of the crystal. As an alternative to X-rays, ultraviolet (UV) radiation has been used to induce specific changes in the macromolecule, which only marginally affects the quality of the diffraction (Nanao and Ravelli 2006) while inducing more selective changes to the protein structure. This method is known as UV-RIP (ultraviolet radiationdamage-induced phasing). The most striking effect of UV radiation damage on protein crystals, as for X-ray radiation, is the breakage of disulfide bonds. Furthermore, this technique has been extended to a non-disulfide-containing protein, photoactive yellow protein, which contains a chromophore covalently attached through a thioester linkage to a cysteine residue (Nanao and Ravelli 2006) and to selenomethionine (MSe) proteins (Panjikar et al. 2011). Therefore, this method offers considerable potential, and selenium-specific UV damage could serve as an additional or even an alternative way of experimental phasing in macromolecular crystallography (de Sanctis et al. 2011). Another popular method adopted at SP centers is the use of iodide ion soaks and SAD experiments for de novo phasing (Abendroth et al. 2011).Fig. 7


Current methods in structural proteomics and its applications in biological sciences
Examples for widely used structure determination methods. a Structure of E. coli Arabinose Isomerase (PDB 2AJT) determined by single-wavelength anomalous diffraction (SAD). Selenomithionine residues are also shown (Manjasetty and Chance 2006). b Structure of DAPK3 (PDB 2J90) determined with the molecular replacement (MR) method using the template prepared by homolog structures (PDB 1YRT, 1JKT, 1WVX) (Pike et al. 2008)
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Related In: Results  -  Collection

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Fig7: Examples for widely used structure determination methods. a Structure of E. coli Arabinose Isomerase (PDB 2AJT) determined by single-wavelength anomalous diffraction (SAD). Selenomithionine residues are also shown (Manjasetty and Chance 2006). b Structure of DAPK3 (PDB 2J90) determined with the molecular replacement (MR) method using the template prepared by homolog structures (PDB 1YRT, 1JKT, 1WVX) (Pike et al. 2008)
Mentions: Heavy-atom incorporation (isomorphous replacement, anomalous scattering and anomalous dispersion), molecular replacement and direct methods are commonly used techniques to solve protein structures. The general requirement for the exploitation of the anomalous signal for the determination of phase estimations via multiple or single-wavelength anomalous diffraction (MAD or SAD) techniques is that the protein crystal should contain anomalously scattering atoms, e.g., Hg, Pt or Se. With the advent of tunable X-ray sources and improved data collection techniques, it is now possible to measure the intensities of diffracted X-rays with very high precision. The small differences in intensities between Bijovoet pairs due to the presence of heavy atoms can be used to calculate initial estimates of the protein phase angle. One of the strategies widely used for the determination of novel protein structures is selenomethionine incorporation, where selenomethionine is replaced by methionine in the protein during expression. This method has revolutionized protein X-ray crystallography and it is estimated that over two-thirds of all novel crystal structures have been determined using either Se-SAD or Se-MAD (Fig. 7a). Novel structures can also be solved using the weak anomalous signals from atoms, such as sulfur and phosphorous present in certain macromolecules. SAD represents the most commonly used technique for novel proteins in SP centers. Multiple or single isomorphous replacement (MIR or SIR) methods also require the introduction of heavy atoms such as mercury, platinum, uranium or gold into the macromolecule under investigation. These heavy atoms must be incorporated into protein crystals without disrupting the lattice interactions so that it remains isomorphous with respect to the native crystal. In the SIR method, intensity differences between the heavy-atom derivatized and native crystal are used to calculate experimental phases. Recently, the SIR phasing protocol has been re-applied in the radiation damage-induced phasing (RIP) technique, where the differences in intensities induced by radiation damage are used as a phasing tool (Ravelli et al. 2003). Limitations of these phasing protocols are mainly due to the deleterious effect that a high X-ray dose has on a protein crystal. X-ray radiation damage induces many changes to the protein structure and to the solvent, resulting in a consistent number of damaged sites and a decrease in the diffraction quality of the crystal. As an alternative to X-rays, ultraviolet (UV) radiation has been used to induce specific changes in the macromolecule, which only marginally affects the quality of the diffraction (Nanao and Ravelli 2006) while inducing more selective changes to the protein structure. This method is known as UV-RIP (ultraviolet radiationdamage-induced phasing). The most striking effect of UV radiation damage on protein crystals, as for X-ray radiation, is the breakage of disulfide bonds. Furthermore, this technique has been extended to a non-disulfide-containing protein, photoactive yellow protein, which contains a chromophore covalently attached through a thioester linkage to a cysteine residue (Nanao and Ravelli 2006) and to selenomethionine (MSe) proteins (Panjikar et al. 2011). Therefore, this method offers considerable potential, and selenium-specific UV damage could serve as an additional or even an alternative way of experimental phasing in macromolecular crystallography (de Sanctis et al. 2011). Another popular method adopted at SP centers is the use of iodide ion soaks and SAD experiments for de novo phasing (Abendroth et al. 2011).Fig. 7

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.