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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.

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.

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Progesterone receptor LBD. Cocrystals of the ligand of interest were obtained when the protein was initially expressed with a lower affinity ligand. The ligand of interest was then added in molar excess and included throughout protein purification.
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fig7: Progesterone receptor LBD. Cocrystals of the ligand of interest were obtained when the protein was initially expressed with a lower affinity ligand. The ligand of interest was then added in molar excess and included throughout protein purification.

Mentions: Since the expression of these nuclear receptors is normally ligand-dependent, what strategy can one use if one is unable to express protein with an important ligand? In such an instance with PR, the protein was expressed in the presence of a low-affinity ligand (∼50 nM). During the cell lysis, protein purification, dialysis and final concentration, a higher affinity ligand of interest (∼5–10 nM) was included in molar excess. This was the only method by which crystals of the ligand of interest were grown (Fig. 7 ▶).


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)

Progesterone receptor LBD. Cocrystals of the ligand of interest were obtained when the protein was initially expressed with a lower affinity ligand. The ligand of interest was then added in molar excess and included throughout protein purification.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Progesterone receptor LBD. Cocrystals of the ligand of interest were obtained when the protein was initially expressed with a lower affinity ligand. The ligand of interest was then added in molar excess and included throughout protein purification.
Mentions: Since the expression of these nuclear receptors is normally ligand-dependent, what strategy can one use if one is unable to express protein with an important ligand? In such an instance with PR, the protein was expressed in the presence of a low-affinity ligand (∼50 nM). During the cell lysis, protein purification, dialysis and final concentration, a higher affinity ligand of interest (∼5–10 nM) was included in molar excess. This was the only method by which crystals of the ligand of interest were grown (Fig. 7 ▶).

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