Fluorescent energy transfer mediated chemical activation (fetma) for the elucidation of the three-dimensional structure of biomacromolecules
AssigneeFraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
InventorsDaniel Hoffmann, Ralf Zimmer
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Abstract
The present invention relates to a method for the selected chemical activation of photo-activatable cross-linker molecules around ligand binding pockets and fluorescent groups in macromolecules, especially biological macromolecules, by using fluorescent ligands of the macromolecule and by selecting photo-activatable cross-linker molecules having specific activation energies so that a radiationless energy transfer (Förster transfer) from the fluorescent ligands to the cross-linker molecules, which become activated thereby, takes place.
Claims7
A method to assist in three-dimensional structural elucidation of a macromolecule, characterized in that
- a) a ligand is introduced into said macromolecule, or its spatial position relative to the macromolecule is determined, said ligand fluorescing upon excitation with a fluorescence frequency within a range of from ν1 to ν2;
- b) one or more photoactivatable bifunctional cross-linkers are covalently bound to functional groups of the macromolecule by reaction with a non-photoactivatable reactive group on the cross-linkers, each cross-linker having a photoactivatable end with an excitation frequency within said range of from ν1 to ν2, and a non-photoactivatable end, wherein said non-photoactivatable end of the cross-linker is covalently bound to the macromolecule;
- c) said macromolecule is irradiated with light having a frequency νQ, which is above the frequency interval ν1 to ν2, wherein the photoactivatable end of the cross-linker is activated for reaction with a surface of the macromolecule by means of radiationless Förster transfer from said ligand to neighboring cross-linkers, which react with the surface of the macromolecule depending on the distance of said ligand from said neigboring cross-linkers, wherein groups linked together in pairs are formed;
- and
- d) groups linked together by said cross-linkers are identified for determination of spatial neighboring relationships between linked groups.
Dependencies are inferred from the claim text.