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: Contribution to journalArticle

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 (1)H 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 (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H 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)
Pages (from-to)47-63
Number of pages17
JournalMethods in molecular biology (Clifton, N.J.)
Volume914
StatePublished - 2012
Externally publishedYes

Fingerprint

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

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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.

In: Methods in molecular biology (Clifton, N.J.), Vol. 914, 2012, p. 47-63.

Research output: Contribution to journalArticle

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. In: Methods in molecular biology (Clifton, N.J.). 2012 ; Vol. 914. pp. 47-63.
@article{d9c263dc8f064436a5f6bd945ccbff92,
title = "Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins.",
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 (1)H 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 (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H 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.",
author = "Venkitakrishnan, {Rani Parvathy} and Outhiriaradjou Benard and Marianna Max and Markley, {John L.} and Assadi-Porter, {Fariba M.}",
year = "2012",
language = "English (US)",
volume = "914",
pages = "47--63",
journal = "Methods in Molecular Biology",
issn = "1064-3745",
publisher = "Humana Press",

}

TY - JOUR

T1 - Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins.

AU - Venkitakrishnan, Rani Parvathy

AU - Benard, Outhiriaradjou

AU - Max, Marianna

AU - Markley, John L.

AU - Assadi-Porter, Fariba M.

PY - 2012

Y1 - 2012

N2 - 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 (1)H 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 (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H 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.

AB - 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 (1)H 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 (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H 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.

UR - http://www.scopus.com/inward/record.url?scp=84872678526&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84872678526&partnerID=8YFLogxK

M3 - Article

C2 - 22976022

VL - 914

SP - 47

EP - 63

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -