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The use of biophysical methods increases success in obtaining liganded crystal structures.

Chung CW - Acta Crystallogr. D Biol. Crystallogr. (2006)

Bottom Line: Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale.Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes.Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural and Biophysical Sciences, GlaxoSmithKline Research and Development, Medicines Research Centre, Gunnelswood Road, Stevenage SG1 2NY, England. cc16943@gsk.com

ABSTRACT
In attempts to determine the crystal structure of small molecule-protein complexes, a common frustration is the absence of ligand binding once the protein structure has been solved. While the first structure, even with no ligand bound (apo), can be a cause for celebration, the solution of dozens of apo structures can give an unwanted sense of déjà vu. Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale. There are many reasons for the lack of observed binding in crystals and this paper highlights some of these. Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes. As there are an overwhelming number of biophysical methods available, some of the factors that need to be considered when choosing the most appropriate technique for a given system are discussed. Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

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(a) Flowchart of a typical protocol to produce a solution of a complex, where protein and ligand are simply combined. (b) The simple combination of protein and ligand may be replaced by an ITC, where additional data on the binding stoichiometry, enthalpy and affinity can be gathered en route to crystallization trials.
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fig2: (a) Flowchart of a typical protocol to produce a solution of a complex, where protein and ligand are simply combined. (b) The simple combination of protein and ligand may be replaced by an ITC, where additional data on the binding stoichiometry, enthalpy and affinity can be gathered en route to crystallization trials.

Mentions: It is possible to envisage how the simplicity and high information content of the ITC experiment, combined with its nondestructive nature, could be incorporated into a cocrystallization protocol where confirmation of complexation is required but protein supply is limited. This is illustrated in Fig. 2 ▶. Fig. 2 ▶(a) shows a ‘typical’ cocrystallization protocol where ligand is added to the protein and the mixture pre-incubated on ice for anything from minutes to hours. Sometimes this is performed at modest protein concentrations so that a concentration step is required and sometimes at higher concentrations. Regardless of the precise details of the process, there are often no checks to confirm the success or completeness of complex formation for small ligands. If this final ‘complex’ solution fails to generate crystals, it is unclear whether this is a complexation issue or a crystallization problem. Alternatively, if only apo crystals are formed, then it is unclear whether any complex was ever present. Fig. 2 ▶(b) shows how the ITC experiment can be used as a more controlled way of combining the protein and ligand, with the additional advantage of generating thermodynamic and binding information ‘for free’. After a successful ITC experiment, where ligand has been injected into a cell containing protein, the final cell contents contain a confirmed saturated protein complex ready to be concentrated for cocrystallization trials.


The use of biophysical methods increases success in obtaining liganded crystal structures.

Chung CW - Acta Crystallogr. D Biol. Crystallogr. (2006)

(a) Flowchart of a typical protocol to produce a solution of a complex, where protein and ligand are simply combined. (b) The simple combination of protein and ligand may be replaced by an ITC, where additional data on the binding stoichiometry, enthalpy and affinity can be gathered en route to crystallization trials.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483471&req=5

fig2: (a) Flowchart of a typical protocol to produce a solution of a complex, where protein and ligand are simply combined. (b) The simple combination of protein and ligand may be replaced by an ITC, where additional data on the binding stoichiometry, enthalpy and affinity can be gathered en route to crystallization trials.
Mentions: It is possible to envisage how the simplicity and high information content of the ITC experiment, combined with its nondestructive nature, could be incorporated into a cocrystallization protocol where confirmation of complexation is required but protein supply is limited. This is illustrated in Fig. 2 ▶. Fig. 2 ▶(a) shows a ‘typical’ cocrystallization protocol where ligand is added to the protein and the mixture pre-incubated on ice for anything from minutes to hours. Sometimes this is performed at modest protein concentrations so that a concentration step is required and sometimes at higher concentrations. Regardless of the precise details of the process, there are often no checks to confirm the success or completeness of complex formation for small ligands. If this final ‘complex’ solution fails to generate crystals, it is unclear whether this is a complexation issue or a crystallization problem. Alternatively, if only apo crystals are formed, then it is unclear whether any complex was ever present. Fig. 2 ▶(b) shows how the ITC experiment can be used as a more controlled way of combining the protein and ligand, with the additional advantage of generating thermodynamic and binding information ‘for free’. After a successful ITC experiment, where ligand has been injected into a cell containing protein, the final cell contents contain a confirmed saturated protein complex ready to be concentrated for cocrystallization trials.

Bottom Line: Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale.Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes.Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural and Biophysical Sciences, GlaxoSmithKline Research and Development, Medicines Research Centre, Gunnelswood Road, Stevenage SG1 2NY, England. cc16943@gsk.com

ABSTRACT
In attempts to determine the crystal structure of small molecule-protein complexes, a common frustration is the absence of ligand binding once the protein structure has been solved. While the first structure, even with no ligand bound (apo), can be a cause for celebration, the solution of dozens of apo structures can give an unwanted sense of déjà vu. Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale. There are many reasons for the lack of observed binding in crystals and this paper highlights some of these. Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes. As there are an overwhelming number of biophysical methods available, some of the factors that need to be considered when choosing the most appropriate technique for a given system are discussed. Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

Show MeSH
Related in: MedlinePlus