Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins

Rani Parvathy Venkitakrishnan, Outhiriaradjou Benard, Marianna Max, John L. Markley, Fariba M. Assadi-Porter

Research output: Chapter in Book/Report/Conference proceedingChapter

24 Citations (Scopus)

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.

Original languageEnglish (US)
Title of host publicationMembrane Protein Structure and Dynamics: Methods and Protocols
PublisherHumana Press Inc.
Pages47-63
Number of pages17
Volume914
ISBN (Print)9781627030229
DOIs
StatePublished - 2012
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume914
ISSN (Print)10643745

Fingerprint

Spectrum Analysis
Membrane Proteins
Ligands
Proteins
Magnetic Resonance Spectroscopy
Sweetening Agents
G-Protein-Coupled Receptors
Names
Proton Magnetic Resonance Spectroscopy

Keywords

  • Dextrose
  • G-protein-coupled receptor (GPCR)
  • Membrane-bound receptors
  • Neotame
  • Saturation transfer difference (STD)
  • Saturation transfer double difference (STDD)
  • Sweet taste receptor
  • T1R2
  • T1R3

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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 Membrane Protein Structure and Dynamics: Methods and Protocols (Vol. 914, pp. 47-63). (Methods in Molecular Biology; Vol. 914). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-23-6_4

Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins. / Venkitakrishnan, Rani Parvathy; Benard, Outhiriaradjou; Max, Marianna; Markley, John L.; Assadi-Porter, Fariba M.

Membrane Protein Structure and Dynamics: Methods and Protocols. Vol. 914 Humana Press Inc., 2012. p. 47-63 (Methods in Molecular Biology; Vol. 914).

Research output: Chapter in Book/Report/Conference proceedingChapter

Venkitakrishnan, RP, Benard, O, Max, M, Markley, JL & Assadi-Porter, FM 2012, Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins. in Membrane Protein Structure and Dynamics: Methods and Protocols. vol. 914, Methods in Molecular Biology, vol. 914, Humana Press Inc., pp. 47-63. https://doi.org/10.1007/978-1-62703-23-6_4
Venkitakrishnan RP, Benard O, Max M, Markley JL, Assadi-Porter FM. Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins. In Membrane Protein Structure and Dynamics: Methods and Protocols. Vol. 914. Humana Press Inc. 2012. p. 47-63. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-62703-23-6_4
Venkitakrishnan, Rani Parvathy ; Benard, Outhiriaradjou ; Max, Marianna ; Markley, John L. ; Assadi-Porter, Fariba M. / Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins. Membrane Protein Structure and Dynamics: Methods and Protocols. Vol. 914 Humana Press Inc., 2012. pp. 47-63 (Methods in Molecular Biology).
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