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Crystallization of protein-ligand complexes.

Hassell AM, An G, Bledsoe RK, Bynum JM, Carter HL, Deng SJ, Gampe RT, Grisard TE, Madauss KP, Nolte RT, Rocque WJ, Wang L, Weaver KL, Williams SP, Wisely GB, Xu R, Shewchuk LM - Acta Crystallogr. D Biol. Crystallogr. (2006)

Bottom Line: Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule.These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form.This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

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Affiliation: Department of Computational, Analytical and Structural Sciences, Glaxo SmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA. annie.m.hassell@gsk.com

ABSTRACT
Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

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Ligand included throughout the protein purification of kinase 1 yielded pure monomeric protein (b). Purification without the ligand resulted in a mixture of protein, lipids and DNA (a).
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fig8: Ligand included throughout the protein purification of kinase 1 yielded pure monomeric protein (b). Purification without the ligand resulted in a mixture of protein, lipids and DNA (a).

Mentions: In one example, a recombinant enzyme (kinase 1) from a baculovirus was expressed in insect cells. The cell lysate was subjected to immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Fig. 8 ▶(a) shows a typical SEC chromatogram for this enzyme. All the protein elutes at 700 ml (the void volume for this column). The enzyme preparation is highly contaminated with other proteins and likely nucleic acids (given the high absorbance at 260 nm). Although this enzyme is active, it is unsuitable for structural studies. In an effort to prepare enzyme for X-ray diffraction studies, an inhibitor specific for the recombinant enzyme was included in the lysis and chromatography buffers. Fig. 8 ▶(b) is the SEC (size-exclusion chromatography) profile of the enzyme plus inhibitor. The proteins eluting at the 700 ml void are the same as seen in Fig. 8 ▶(a), but eluting at about 950 ml (the position expected for monomeric enzyme) is enzyme that is homogenous. The enzyme purified in the presence of inhibitor was successfully crystallized, enabling the three-dimensional structure to be determined.


Crystallization of protein-ligand complexes.

Hassell AM, An G, Bledsoe RK, Bynum JM, Carter HL, Deng SJ, Gampe RT, Grisard TE, Madauss KP, Nolte RT, Rocque WJ, Wang L, Weaver KL, Williams SP, Wisely GB, Xu R, Shewchuk LM - Acta Crystallogr. D Biol. Crystallogr. (2006)

Ligand included throughout the protein purification of kinase 1 yielded pure monomeric protein (b). Purification without the ligand resulted in a mixture of protein, lipids and DNA (a).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483499&req=5

fig8: Ligand included throughout the protein purification of kinase 1 yielded pure monomeric protein (b). Purification without the ligand resulted in a mixture of protein, lipids and DNA (a).
Mentions: In one example, a recombinant enzyme (kinase 1) from a baculovirus was expressed in insect cells. The cell lysate was subjected to immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Fig. 8 ▶(a) shows a typical SEC chromatogram for this enzyme. All the protein elutes at 700 ml (the void volume for this column). The enzyme preparation is highly contaminated with other proteins and likely nucleic acids (given the high absorbance at 260 nm). Although this enzyme is active, it is unsuitable for structural studies. In an effort to prepare enzyme for X-ray diffraction studies, an inhibitor specific for the recombinant enzyme was included in the lysis and chromatography buffers. Fig. 8 ▶(b) is the SEC (size-exclusion chromatography) profile of the enzyme plus inhibitor. The proteins eluting at the 700 ml void are the same as seen in Fig. 8 ▶(a), but eluting at about 950 ml (the position expected for monomeric enzyme) is enzyme that is homogenous. The enzyme purified in the presence of inhibitor was successfully crystallized, enabling the three-dimensional structure to be determined.

Bottom Line: Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule.These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form.This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Computational, Analytical and Structural Sciences, Glaxo SmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA. annie.m.hassell@gsk.com

ABSTRACT
Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

Show MeSH