Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-14
2004-03-30
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007900, C435S007920, C435S174000, C435S968000, C435S973000, C436S002000, C436S056000, C436S057000, C436S063000, C436S164000, C436S172000, C436S514000, C436S518000, C436S800000, C436S804000, C436S805000, C436S819000, C530S300000, C530S350000, C250S281000
Reexamination Certificate
active
06713256
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
The method according to the invention is preferably used to focus a per se known bioanalytical method for obtaining information about the three-dimensional structure of biomacromolecules to functionally relevant portions of biomacromolecules, such as ligand binding pockets. In this bioanalytical method, the execution of the method according to the invention is followed by: specific digestion of the biomacromolecule, separation of its components by chromatography and mass spectrometry, and a computer simulation for obtaining three-dimensional structural models with the experimentally obtained information as a boundary condition. For refining the structure, the whole procedure may be run in several iterations.
2. Review of the Related Art
Biological macromolecules, such as proteins, ribonucleic acids or macromolecular complexes of different biopolymers, such as ribosomes, are the carriers of the essential biochemical functions of almost all vital processes in biological organisms. Generally, these functions depend on exactly defined three-dimensional structures of the biomacromolecules. From the knowledge of a three-dimensional structure, the functional mechanism can be concluded. This makes three-dimensional structures of biological macromolecules an important source of information for molecular medicine and pharmacology.
Particularly valuable is information about the three-dimensional structure of the binding pockets of biological macromolecules since they are the proper functionally important portions of the macromolecules, because this is where interactions with other, bound molecules, such as ligands or substrates, take place. One of a large number of medicinally relevant examples of how the knowledge of the three-dimensional structure of binding pockets is utilized is the so-called rational design of inhibitors of viral or bacterial enzymes. These inhibitors are designed in such a way that they fit into the binding pockets of the enzymes like keys into locks. Thus, the enzymes become blocked, and the reproduction of the viruses or bacteria is stopped. Such a selective key-lock design is possible only when the three-dimensional structure of the binding pocket is known.
In the prior art, the determination of the three-dimensional structures of biological macromolecules using conventional methods such as X-ray crystallographic analysis or nuclear magnetic resonance has generally been difficult and time-consuming. This has many reasons. In particular, these methods require large (typically millimolar) amounts of the purified macromolecule in a special form, either crystalline or as a concentrated solution. In addition, for solving the three-dimensional structure, further difficulties must be overcome, such as the solution of the so-called phase problem in crystallography. Thus, in total, the elucidation of the three-dimensional structure of a biomacromolecule can take several person-years.
SUMMARY OF THE INVENTION
It has been the object of the invention to provide a method for three-dimensional structural elucidation which requires small amounts of pure macromolecule and can be focused on functionally relevant binding pockets. In addition, the method according to the invention is to be applicable in cases which are particularly difficult to treat with conventional methods. These include, for example, membrane proteins and large globular proteins and complexes which are difficult to purify and difficult to crystallize.
In a first embodiment, the method according to the invention for the three-dimensional structural elucidation of macromolecules (M) is characterized in that a ligand (F) capable of fluorescence with a fluorescence frequency within a range of from &ngr;
1
to &ngr;
2
is introduced into said macromolecule (M), or its spatial position relative to the macromolecule (M) is determined by per se known methods; one or more photoactivatable bifunctional cross-linkers C, C′, C″ with their respective excitation frequencies within the range of from &ngr;
1
to &ngr;
2
are covalently bound between the non-photoactivatable end S of the cross-linker C, C′, C″ and suitable functional groups m of the macromolecule M with exclusion of light; said macromolecule M is irradiated above the frequency interval &ngr;
1
to &ngr;
2
with a frequency &ngr;
Q
wherein the photoactivatable end A and/or A′ of the cross-linker C and/or C″ is activated for reaction with the surface of macromolecule M by means of radiationless transfer (Förster transfer) to neighboring cross-linkers C and/or C″, and reacts with the surface of macromolecule M depending on the distance of said ligand (F) capable of fluorescence; and the groups linked together in pairs are identified by bioanalytical methods, especially specific digestion of macromolecule M, the digestion fragments are separated, especially by their mass, and spatial neighboring relationships are determined by calculation.
Further embodiments of the invention can be seen, in particular, from the dependent claims.
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Hoffmann Daniel
Zimmer Ralf
Brobeck Phleger & Harrison LLP
Fraunhofer Gesellschaft zur Förderung der angewandten Forschung
Le Long V.
Padmanabhan Kartic
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