Fluorescent energy transfer mediated chemical activation (fetma) for the elucidation of the three-dimensional structure of biomacromolecules

• US 6,713,256 B1
• Filed: 08/14/2000
• Issued: 03/30/2004
• Est. Priority Date: 02/14/1998
• Status: Expired due to Fees

• Current Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Original Assignee: Fraunhofer Gesellschaft Zur Foerderung Der Angewandten Forsching E.V.
• Inventors: Hoffmann, Daniel, Zimmer, Ralf
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Padmanabhan, Kartic
• Application Number:
  US09/622,161
• Time in Patent Office: 1,324 Days
• Field of Search: 435/6, 435/7.1, 435/7.9, 435/7.92, 435/174, 435/968, 435/973, 436/56, 436/63, 436/164, 436/172, 436/805, 436/514, 436/518, 436/2, 436/57, 436/800, 436/804, 436/819, 530/300, 530/350, 250/281
• US Class Current: 435/6.14
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody
• CPC Class Codes: Y10T 436/13 Tracers or tags

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.

Claims

1. 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 ν

₁ to ν

₂;

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 ν

₁ to ν

₂, 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 ν

_Q, which is above the frequency interval ν

₁ to ν

₂, wherein the photoactivatable end of the cross-linker is activated for reaction with a surface of the macromolecule by means of radiationless Fö

rster 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.

2. The method according to claim 1, characterized in that said macromolecules are selected from proteins, ribonucleic acids or macromolecular complexes of different biopolymers.

3. The method according to claim 1, characterized in that direct photoactivation of cross-linkers occurs simultaneously with reaction of bifunctional cross-linkers binding to the macromolecule with chemical specificity.

4. The method according to claim 1, characterized in that 4-[p-azidosalicylamido]butylamine is used as the cross-linker.

5. The method according to claim 1, wherein said groups linked in pairs are identified by bioanalytical methods comprising specific digestion of the macromolecule, and digestion fragments are separated.

6. The method according to claim 5, characterized in that the digestion fragments are separated by their masses.

7. The method according to any one of claims 1-6, characterized in that the method is repeated several times, optionally using different ligands, cross-linkers and/or digestion reagents.