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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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Effect of the F602S mutation on GR expression. Comparison of the protein expression of wild-type GR (lane 1) and F602S GR (lane 2) in the presence of 10 µM dexamethasone. The proteins shown are the soluble fractions from the Ni2+ column. Lanes 3–5 show the purification of the F602S LBD (lane 3, sample after thrombin digestion; lane 4, Ni2+ column flowthrough of the thrombin-digested material; lane 5, final purified protein; Bledsoe et al., 2002 ▶).
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fig2: Effect of the F602S mutation on GR expression. Comparison of the protein expression of wild-type GR (lane 1) and F602S GR (lane 2) in the presence of 10 µM dexamethasone. The proteins shown are the soluble fractions from the Ni2+ column. Lanes 3–5 show the purification of the F602S LBD (lane 3, sample after thrombin digestion; lane 4, Ni2+ column flowthrough of the thrombin-digested material; lane 5, final purified protein; Bledsoe et al., 2002 ▶).

Mentions: These techniques were still not sufficient to provide soluble protein for the recalcitrant nuclear receptors GR and MR. Expression studies with GR demonstrated that mutating the phenylalanine at position 602 to a serine residue (F602S), as in PR, led to increased expression of this receptor (Bledsoe et al., 2002 ▶; Fig. 2 ▶).


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)

Effect of the F602S mutation on GR expression. Comparison of the protein expression of wild-type GR (lane 1) and F602S GR (lane 2) in the presence of 10 µM dexamethasone. The proteins shown are the soluble fractions from the Ni2+ column. Lanes 3–5 show the purification of the F602S LBD (lane 3, sample after thrombin digestion; lane 4, Ni2+ column flowthrough of the thrombin-digested material; lane 5, final purified protein; Bledsoe et al., 2002 ▶).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Effect of the F602S mutation on GR expression. Comparison of the protein expression of wild-type GR (lane 1) and F602S GR (lane 2) in the presence of 10 µM dexamethasone. The proteins shown are the soluble fractions from the Ni2+ column. Lanes 3–5 show the purification of the F602S LBD (lane 3, sample after thrombin digestion; lane 4, Ni2+ column flowthrough of the thrombin-digested material; lane 5, final purified protein; Bledsoe et al., 2002 ▶).
Mentions: These techniques were still not sufficient to provide soluble protein for the recalcitrant nuclear receptors GR and MR. Expression studies with GR demonstrated that mutating the phenylalanine at position 602 to a serine residue (F602S), as in PR, led to increased expression of this receptor (Bledsoe et al., 2002 ▶; Fig. 2 ▶).

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