Energetics by NMR: Site-Specific Binding in a Positively Cooperative System

Proteins with multiple binding sites exhibit a complex behavior that depends on the intrinsic affinities for each site and the energetic communication between the sites. The contributions from intrinsic affinity and cooperativity are difficult to deconvolute using conventional binding experiments th...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2002-02, Vol.99 (4), p.1847-1852
Main Authors: Tochtrop, Gregory P., Richter, Klaus, Tang, Changguo, Toner, James J., Covey, Douglas F., Cistola, David P.
Format: Article
Language:English
Subjects:
Allosteric Site
Bayes Theorem
Bile acids
Binding Sites
Biochemistry
Biological Sciences
Calorimetry
Carrier proteins
Carrier Proteins - chemistry
Dose-Response Relationship, Drug
Escherichia coli - metabolism
Free energy
Glycocholic Acid - chemistry
Glycocholic Acid - pharmacology
Heat measurement
Humans
Hydroxysteroid Dehydrogenases
Ileum - chemistry
Isotherms
Kinetics
Ligands
Magnetic Resonance Spectroscopy - methods
Membrane Glycoproteins
Models, Chemical
Molecular biology
NMR
Nuclear magnetic resonance
Protein Binding
Proteins
Recombinant Proteins - metabolism
Spectral correlation
Temperature
Thermodynamics
Time Factors
Titration
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Summary:Proteins with multiple binding sites exhibit a complex behavior that depends on the intrinsic affinities for each site and the energetic communication between the sites. The contributions from intrinsic affinity and cooperativity are difficult to deconvolute using conventional binding experiments that lack information about the occupancies of individual sites. Here, we report the concerted use of NMR and isothermal titration calorimetry to determine the intrinsic and cooperative binding free energies for a ligand-protein complex. The NMR measurements provided the site-specific information necessary to resolve the binding parameters. Using this approach, we observed that human ileal bile acid binding protein binds two molecules of glycocholic acid with low intrinsic affinity but an extraordinarily high degree of positive cooperativity. The highly cooperative nature of the binding provides insights into the protein's biological mechanism. With ongoing improvements in sensitivity and resolution, NMR methods are becoming more amenable to dissecting the complex binding energetics of multisite systems.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.012379199