Use of a distant reporter group as evidence for a conformational change in a sensory receptor

R. Suzanne Zukin, P. R. Hartig, D. E. Koshland

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of both the fluorescein and tryptophan, as shown by both spectral and potassium iodide quenching changes. The distance between the two dyes is established by fluorescence energy transfer methods to be 41 ± 10 Å. Since only one molecule of galactose binds per molecule of receptor and since the galactose molecule is only about 5 Å in length, changes at one of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 Å through the receptor molecule.

Original languageEnglish (US)
Pages (from-to)1932-1936
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume74
Issue number5
StatePublished - 1977
Externally publishedYes

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Sensory Receptor Cells
Ligands
Galactose
Tryptophan
Potassium Iodide
Energy Transfer
Salmonella typhimurium
Fluorescein
Methionine
Catalytic Domain
Coloring Agents
Fluorescence
Proteins
galactose receptor

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Use of a distant reporter group as evidence for a conformational change in a sensory receptor. / Zukin, R. Suzanne; Hartig, P. R.; Koshland, D. E.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 74, No. 5, 1977, p. 1932-1936.

Research output: Contribution to journalArticle

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N2 - A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of both the fluorescein and tryptophan, as shown by both spectral and potassium iodide quenching changes. The distance between the two dyes is established by fluorescence energy transfer methods to be 41 ± 10 Å. Since only one molecule of galactose binds per molecule of receptor and since the galactose molecule is only about 5 Å in length, changes at one of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 Å through the receptor molecule.

AB - A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of both the fluorescein and tryptophan, as shown by both spectral and potassium iodide quenching changes. The distance between the two dyes is established by fluorescence energy transfer methods to be 41 ± 10 Å. Since only one molecule of galactose binds per molecule of receptor and since the galactose molecule is only about 5 Å in length, changes at one of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 Å through the receptor molecule.

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