科研之友
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摘要
Tryptophan-induced quenching of fluorophores (TrIQ) uses intramolecular fluorescence quenching to assess distances in proteins too small (%26lt;15 angstrom) 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 %26quot;static quenching%26quot; (contact between the Tip 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 Trill tyrosine-induced quenching (TyrIQ) occurs for the fluorophore bimane in a distance-dependent fashion, although with some key differences. The Tyr %26quot;sphere of quenching%26quot; for birnane (%26lt;= 10 angstrom) is smaller than for Tip (%26lt;= 15 angstrom, C alpha-C alpha distance), and the size difference between the quenching residue (Tyr) and control (Phe) differs by only a hydroxyl group. Second, we show how Trill and TyrIQ can be used together to assess the magnitude and energetics of a protein movement. In these studies, we placed a birnane (probe) and Tip or Tyr (quencher) on opposite ends of a %26quot;hinge%26quot; in T4L and conducted TrIQ and TyrIQ measurements. Our results are consistent with an similar to 5 angstrom change in C alpha-C alpha distances between these sites upon substrate binding, in agreement with the crystal structures. Subsequent Arrhenius analysis suggests the activation energy barrier (E-a) to this movement is relatively low (similar to 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.
- 出版日期2014-10-14
