2v3l
ORIENTATIONAL AND DYNAMICAL HETEROGENEITY OF RHODAMINE 6G TERMINALLY ATTACHED TO A DNA HELIX
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OverviewOverview
The comparison of Forster resonance energy transfer (FRET) efficiencies between two fluorophores covalently attached to a single protein or DNA molecule is an elegant approach for deducing information about their structural and dynamical heterogeneity. For a more detailed structural interpretation of single-molecule FRET assays, information about the positions as well as the dynamics of the dye labels attached to the biomolecule is important. In this work, Rhodamine 6G (2-[3'-(ethylamino)-6'-(ethylimino)-2',7'-dimethyl-6'H-xanthen-9'-yl]-benz oic acid) bound to the 5'-end of a 20 base pair long DNA duplex is investigated by both single-molecule multiparameter fluorescence detection (MFD) experiments and NMR spectroscopy. Rhodamine 6G is commonly employed in nucleic acid research as a FRET dye. MFD experiments directly reveal the equilibrium of the dye bound to DNA between three heterogeneous environments, which are characterized by distinct fluorescence lifetime and intensity distributions as a result of different guanine-dye excited-state electron transfer interactions. Sub-ensemble fluorescence autocorrelation analysis shows the highly dynamic character of the dye-DNA interactions ranging from nano- to milliseconds and species-specific triplet relaxation times. Two-dimensional NMR spectroscopy corroborates this information by the determination of the detailed geometric structures of the dye-nucleobase complex and their assignment to each population observed in the single-molecule fluorescence experiments. From both methods, a consistent and detailed molecular description of the structural and dynamical heterogeneity is obtained.
About this StructureAbout this Structure
2V3L is a Single protein structure of sequence from [1]. Full crystallographic information is available from OCA.
ReferenceReference
Orientational and dynamical heterogeneity of rhodamine 6G terminally attached to a DNA helix revealed by NMR and single-molecule fluorescence spectroscopy., Neubauer H, Gaiko N, Berger S, Schaffer J, Eggeling C, Tuma J, Verdier L, Seidel CA, Griesinger C, Volkmer A, J Am Chem Soc. 2007 Oct 24;129(42):12746-55. Epub 2007 Sep 27. PMID:17900110
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