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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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A protein concentration of 1 mg ml−1 during complex formation and cross-seeding were critical to obtaining cocrystals of the kinase 4 complexes.
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fig12: A protein concentration of 1 mg ml−1 during complex formation and cross-seeding were critical to obtaining cocrystals of the kinase 4 complexes.

Mentions: Sometimes it is possible to concentrate our protein and then add the ligand to form the complex. However, if the ligand is insoluble, it may cause the protein to precipitate when it is at higher concentrations. It may be necessary to add dilute ligand to diluted protein to achieve good ligand binding with these very insoluble compounds. Kinase 4 had to be diluted to 1 mg ml−1 and then complexed with dilute ligand at a 1:3 protein:ligand ratio to achieve a stable complex that yielded well diffracting crystals (Fig. 12 ▶). The majority of these complex cocrystals were grown by cross-seeding using apo crystals.


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)

A protein concentration of 1 mg ml−1 during complex formation and cross-seeding were critical to obtaining cocrystals of the kinase 4 complexes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig12: A protein concentration of 1 mg ml−1 during complex formation and cross-seeding were critical to obtaining cocrystals of the kinase 4 complexes.
Mentions: Sometimes it is possible to concentrate our protein and then add the ligand to form the complex. However, if the ligand is insoluble, it may cause the protein to precipitate when it is at higher concentrations. It may be necessary to add dilute ligand to diluted protein to achieve good ligand binding with these very insoluble compounds. Kinase 4 had to be diluted to 1 mg ml−1 and then complexed with dilute ligand at a 1:3 protein:ligand ratio to achieve a stable complex that yielded well diffracting crystals (Fig. 12 ▶). The majority of these complex cocrystals were grown by cross-seeding using apo crystals.

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