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Method Article
Giorgio Pochetti
Istituto di Cristallografia, CNR, Montelibretti, 00015 Monterotondo Stazione, Roma, ITALY
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
It could be very useful in drug development to determine the relative affinities of a ligand that can bind to a protein with two (or more) different and specific binding sites.
X-ray crystallography is a very powerful technique to determine the locations and describe the features of each binding site but it is unuseful to establish their relative affinities. Moreover, the stoichiometry of binding observed in the crystal structure of a complex could be an artifact of the crystallization conditions.
On the contrary, isothermal titration calorimetry (ITC) can be used to assign the correct stoichiometry of the association, depending on the initial and final concentration ratio between ligand and protein, and to establish the affinity for each specific binding site, if combined with structural information from X-ray crystallography.
In this protocol a direct and reverse titration is performed to determine the stoichiometry of the ligand binding. A third titration is necessary, after pre-equilibration of the protein with another ligand that is known (from X-ray crystallography) to bind univocally to only one out of the two sites, to assign the association constants for each site.
Keywords
ITC, microcalorimetry, X-ray crystallography, association constant, protein binding site, reverse titration
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The authors declare no competing financial interests
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Associated Publications
New 2-Aryloxy-3-phenyl-propanoic Acids As Peroxisome Proliferator-Activated Receptors alpha/gamma Dual Agonists Able to Upregulate the Mitochondrial Carnitine Shuttle System Gene Expression
by Antonio Laghezza, Giorgio Pochetti, Antonio Lavecchia, +10
Journal Of Medicinal Chemistry (18 December, 2012)
Method Article
Giorgio Pochetti
Istituto di Cristallografia, CNR, Montelibretti, 00015 Monterotondo Stazione, Roma, ITALY
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 20 Dec, 2012
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
It could be very useful in drug development to determine the relative affinities of a ligand that can bind to a protein with two (or more) different and specific binding sites.
X-ray crystallography is a very powerful technique to determine the locations and describe the features of each binding site but it is unuseful to establish their relative affinities. Moreover, the stoichiometry of binding observed in the crystal structure of a complex could be an artifact of the crystallization conditions.
On the contrary, isothermal titration calorimetry (ITC) can be used to assign the correct stoichiometry of the association, depending on the initial and final concentration ratio between ligand and protein, and to establish the affinity for each specific binding site, if combined with structural information from X-ray crystallography.
In this protocol a direct and reverse titration is performed to determine the stoichiometry of the ligand binding. A third titration is necessary, after pre-equilibration of the protein with another ligand that is known (from X-ray crystallography) to bind univocally to only one out of the two sites, to assign the association constants for each site.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Loading...
Associated Publications
New 2-Aryloxy-3-phenyl-propanoic Acids As Peroxisome Proliferator-Activated Receptors alpha/gamma Dual Agonists Able to Upregulate the Mitochondrial Carnitine Shuttle System Gene Expression
by Antonio Laghezza, Giorgio Pochetti, Antonio Lavecchia, +10
Journal Of Medicinal Chemistry (18 December, 2012)