Abstract
Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a 1H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein 1H NMR signals. 1H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the 1H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Wang YS et al (2004) Competition STD NMR for the detection of high-affinity ligands and NMR-based screening. Magn Reson Chem 42:485–489
Assadi-Porter FM et al (2008) Direct NMR detection of the binding of functional ligands to the human sweet receptor, a heterodimeric family 3 GPCR. J Am Chem Soc 130:7212–7213
Mayer MMayer B (1999) Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Ed 38:1784–1788
Assadi-Porter FM et al (2010) Interactions between the human sweet-sensing T1R2-T1R3 receptor and sweeteners detected by saturation transfer difference NMR spectroscopy. Biochim Biophys Acta 1798:82–86
Balaram P et al (1972) Negative nuclear Overhauser effects as probes of macromolecular structure. J Am Chem Soc 94:4015–4017
Megy S et al (2005) STD and TRNOESY NMR studies on the conformation of the oncogenic protein beta-catenin containing the phosphorylated motif DpSGXXpS bound to the beta-TrCP protein. J Biol Chem 280:29107–29116
Claasen B et al (2005) Direct observation of ligand binding to membrane proteins in living cells by a saturation transfer double difference (STDD) NMR spectroscopy method shows a significantly higher affinity of integrin alpha(IIb)beta3 in native platelets than in liposomes. J Am Chem Soc 127:916–919
Mari S et al (2004) 1D saturation transfer difference NMR experiments on living cells: the DC-SIGN/oligomannose interaction. Angew Chem Int Ed Engl 44:296–298
Di Micco S et al (2005) Differential-frequency saturation transfer difference NMR spectroscopy allows the detection of different ligand-DNA binding modes. Angew Chem Int Ed Engl 45:224–228
Angulo J et al (2010) Ligand-receptor binding affinities from saturation transfer difference (STD) NMR spectroscopy: the binding isotherm of STD initial growth rates. Chemistry 16:7803–7812
Feher K et al (2008) Competition saturation transfer difference experiments improved with isotope editing and filtering schemes in NMR-based screening. J Am Chem Soc 130:17148–17153
Assadi-Porter FM et al (2010) Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor. J Mol Biol 398:584–599
Lowry OH et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Cui M et al (2006) The heterodimeric sweet taste receptor has multiple potential ligand binding sites. Curr Pharm Des 12:4591–4600
Jiang P et al (2005) Lactisole interacts with the transmembrane domains of human T1R3 to inhibit sweet taste. J Biol Chem 280:15238–15246
Jiang P et al (2004) The cysteine-rich region of T1R3 determines responses to intensely sweet proteins. J Biol Chem 279:45068–45075
Acknowledgement
This research was supported by NIH grants R21 DC008805 to MM and FAP, R01 DC009018 and Wisconsin Institute of Discovery Grant (WID-135A039) to FAP, R01 DC006696, R01 DC008301 to MM, and P41 RR02301 which funds the National Magnetic Resonance Facility at Madison. We thank Drs. Marco Tonelli and Roger Chylla for their helpful discussions and assistance with the Newton program developed by Dr. Chylla. We thank the NutraSweet Company for providing the sample of neotame.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Venkitakrishnan, R.P., Benard, O., Max, M., Markley, J.L., Assadi-Porter, F.M. (2012). Use of NMR Saturation Transfer Difference Spectroscopy to Study Ligand Binding to Membrane Proteins. In: Vaidehi, N., Klein-Seetharaman, J. (eds) Membrane Protein Structure and Dynamics. Methods in Molecular Biology, vol 914. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-023-6_4
Download citation
DOI: https://doi.org/10.1007/978-1-62703-023-6_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-022-9
Online ISBN: 978-1-62703-023-6
eBook Packages: Springer Protocols