Distance mapping in proteins using fluorescence spectroscopy: Tyrosine, like tryptophan, quenches bimane fluorescence in a distance-dependent manner

Amber M. Jones Brunette, David L. Farrens

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Tryptophan-induced quenching of fluorophores (TrIQ) uses intramolecular fluorescence quenching to assess distances in proteins too small (<15 Å) to be easily probed by traditional Forster resonance energy transfer methods. A powerful aspect of TrIQ is its ability to obtain an ultrafast snapshot of a protein conformation, by identifying "static quenching" (contact between the Trp and probe at the moment of light excitation). Here we report new advances in this site-directed fluorescence labeling (SDFL) approach, gleaned from recent studies of T4 lysozyme (T4L). First, we show that like TrIQ, tyrosine-induced quenching (TyrIQ) occurs for the fluorophore bimane in a distance-dependent fashion, although with some key differences. The Tyr "sphere of quenching" for bimane (≤10 Å) is smaller than for Trp (≤15 Å, Cα-Cα distance), and the size difference between the quenching residue (Tyr) and control (Phe) differs by only a hydroxyl group. Second, we show how TrIQ and TyrIQ can be used together to assess the magnitude and energetics of a protein movement. In these studies, we placed a bimane (probe) and Trp or Tyr (quencher) on opposite ends of a "hinge" in T4L and conducted TrIQ and TyrIQ measurements. Our results are consistent with an ∼5 Å change in Cα-Cα distances between these sites upon substrate binding, in agreement with the crystal structures. Subsequent Arrhenius analysis suggests the activation energy barrier (Ea) to this movement is relatively low (∼1.5-2.5 kcal/mol). Together, these results demonstrate that TyrIQ, used together with TrIQ, significantly expands the power of quenching-based distance mapping SDFL studies.

Original languageEnglish (US)
Pages (from-to)6290-6301
Number of pages12
JournalBiochemistry
Volume53
Issue number40
DOIs
StatePublished - Oct 14 2014

ASJC Scopus subject areas

  • Biochemistry

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