title: Distance mapping in proteins using fluorescence spectroscopy: tyrosine, like tryptophan, quenches bimane fluorescence in a distance-dependent manner.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University , Portland, Oregon 97239-3098, United States.
authors.forename: Amber M
authors.initials: AM
authors.lastname: Jones Brunette
authors.forename: David L
authors.initials: DL
authors.lastname: Farrens
mesh_terms: D001896:Boron Compounds / Q000737:chemistry; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry*; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005456:Fluorescent Dyes; D010457:Peptidoglycan; C021149:bimanes; D014443:Tyrosine; D014364:Tryptophan; D009113:Muramidase