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.
Site-specific free energy changes in proteins upon ligand binding by NMR: Ca(2+) -displacement by Ln(3+) in a Ca(2+) -binding protein from Entamoeba histolytica.
Site-specific free energy changes in proteins upon ligand binding by NMR: Ca(2+) -displacement by Ln(3+) in a Ca(2+) -binding protein from Entamoeba histolytica.
Site-specific free energy changes in proteins upon ligand binding by NMR: Ca(2+) -displacement by Ln(3+) in a Ca(2+) -binding protein from Entamoeba histolytica.
Chem Biol Drug Des. 2011 Jan 14;
Authors: Chandra K, Mustafi SM, Muthukumar S, Chary KV
The study of protein-ligand interaction has been of a great interest in contemporary structural biology. The understanding of the nature...
nmrlearner
Journal club
0
01-18-2011 10:22 PM
[NMR paper] NMR study on the binding of d(GGAAATTTCC)2 with a positively charged pentacosapeptide
NMR study on the binding of d(GGAAATTTCC)2 with a positively charged pentacosapeptide.
Related Articles NMR study on the binding of d(GGAAATTTCC)2 with a positively charged pentacosapeptide.
Biochim Biophys Acta. 1998 Nov 8;1442(2-3):137-47
Authors: van Lieshout E, Hemminga MA
To obtain a better understanding of the electrostatic nature of protein-nucleic acid interactions, we have investigated the interaction of a double-stranded decamer d(GGAAATTTCC)2 with a synthetic arginine and lysine-rich pentacosapeptide (Pep25), using NMR and optical...
nmrlearner
Journal club
0
11-17-2010 11:15 PM
[NMR paper] A residue-specific NMR view of the non-cooperative unfolding of a molten globule.
A residue-specific NMR view of the non-cooperative unfolding of a molten globule.
Related Articles A residue-specific NMR view of the non-cooperative unfolding of a molten globule.
Nat Struct Biol. 1997 Aug;4(8):630-4
Authors: Schulman BA, Kim PS, Dobson CM, Redfield C
Molten globules are partially folded forms of proteins that are thought to be general intermediates in protein folding. Nonetheless, there is limited structural information about such species because they possess conformational heterogeneity and complex dynamical properties that...
nmrlearner
Journal club
0
08-22-2010 05:08 PM
[NMR paper] NMR analysis of site-specific ligand binding in oligomeric proteins. Dynamic studies
NMR analysis of site-specific ligand binding in oligomeric proteins. Dynamic studies on the interaction of riboflavin synthase with trifluoromethyl-substituted intermediates.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles NMR analysis of site-specific ligand binding in oligomeric proteins. Dynamic studies on the interaction of riboflavin synthase with trifluoromethyl-substituted intermediates.
Biochemistry. 1996 Jul 30;35(30):9637-46
Authors: Scheuring J, Fischer M, Cushman M, Lee J, Bacher A,...
nmrlearner
Journal club
0
08-22-2010 02:20 PM
[NMR paper] A constraint reasoning system for automating sequence-specific resonance assignments
A constraint reasoning system for automating sequence-specific resonance assignments from multidimensional protein NMR spectra.
Related Articles A constraint reasoning system for automating sequence-specific resonance assignments from multidimensional protein NMR spectra.
Proc Int Conf Intell Syst Mol Biol. 1993;1:447-55
Authors: Zimmerman DE, Kulikowski CA, Montelione GT
AUTOASSIGN is a prototype expert system designed to aid in the determination of protein structure from nuclear magnetic resonance (NMR) measurements. In this paper we focus...
nmrlearner
Journal club
0
08-21-2010 11:53 PM
[NMR paper] Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-N
Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
Related Articles Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of Trp 62-lysozyme.
J Biochem. 1991 Aug;110(2):295-300
Authors: Nakazawa T, Sakiyama F
The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed...
Energetics by NMR: site-specific binding in a positively cooperative system.
Linear Mode
