EP1057020A2 - Chemical activation mediated by the transfer of fluorescent energy for elucidating the 3-d structure of biological macromolecules - Google Patents

Chemical activation mediated by the transfer of fluorescent energy for elucidating the 3-d structure of biological macromolecules

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Publication number
EP1057020A2
EP1057020A2 EP99911675A EP99911675A EP1057020A2 EP 1057020 A2 EP1057020 A2 EP 1057020A2 EP 99911675 A EP99911675 A EP 99911675A EP 99911675 A EP99911675 A EP 99911675A EP 1057020 A2 EP1057020 A2 EP 1057020A2
Authority
EP
European Patent Office
Prior art keywords
macromolecule
cross
photoactivatable
groups
frequency
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99911675A
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German (de)
French (fr)
Inventor
Daniel Hoffmann
Ralf Dr. Zimmer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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GMD Forschungszentrum Informationstechnik GmbH
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Publication of EP1057020A2 publication Critical patent/EP1057020A2/en
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/973—Simultaneous determination of more than one analyte
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/80—Fluorescent dyes, e.g. rhodamine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/804—Radioisotope, e.g. radioimmunoassay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/805—Optical property
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/819—Multifunctional antigen or antibody
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/13—Tracers or tags

Definitions

  • FLUORESCENCE ENERGY TRANSFER OF MEDIATED CHEMICAL ACTIVATION (FETMA) FOR THE ENLARGEMENT OF THE 3D STRUCTURE OF
  • the invention relates to a method for the targeted chemical activation of photo-activatable cross-linker molecules around ligand binding pockets and fluorescent groups in macromolecules, in particular biological macromolecules, by using fluorescent ligands of the macromolecule and by selecting photo-activatable cross-linker molecules with specific activation energies. so that radiation-free energy transfer (Förster transfer) from the fluorescent ligands to the crosslinker molecules activated thereby takes place.
  • radiation-free energy transfer Förster transfer
  • the method according to the invention is preferably used to focus a bioanalytical method known per se for obtaining information about the SD structure of biomacromolecules on functionally relevant parts of biomacromolecules such as ligand binding pockets.
  • This bioanalytical method is followed by the execution of the method according to the invention: a specific digestion of the biomacromolecule, a separation of the components by chromatography and mass spectrometry, and a computer simulation to obtain SD structure models with the experimentally obtained information as a boundary condition.
  • the entire process can be carried out in several iterations to refine the structure.
  • Biomacromolecules such as proteins, ribonucleic acids or macromolecular complexes from various biopolymers such as ribosomes are the carriers of essential biochemical functions for almost all life processes in biological organisms. In general, these functions are linked to the precisely defined 3D structures of the biomacromolecules. If a 3D structure is known, the functional mechanism can be concluded. This makes 3D biological structures an important source of information for molecular medicine and pharmacology. Information about the 3D structure of the binding pockets of biological macromolecules is particularly valuable because they are the actually functionally important parts of the macromolecules, because they interact with other bound molecules such as ligands or substrates.
  • inventive method should be applicable in cases that are particularly difficult with conventional. Methods to be treated. These include, for example, membrane proteins as well as large, difficult to clean and difficult to crystallize globular proteins and complexes.
  • the method according to the invention for 3D structure elucidation of macromolecules is characterized in that a fluorescent ligand (F) with a fluorescence frequency in the range from v to v 2 is introduced into the macromolecule (M) or its spatial position determined to the macromolecule (M) by methods known per se, one or more photoactivatable bifunctional cross-linkers C, C ⁇ C "with a respective excitation frequency in the range from v to v 2 covalently between the non-photoactivatable end S of the cross-linker C, C ⁇ C "and suitable functional groups m of the macromolecule M binds with the exclusion of light, the macromolecule M is irradiated above the frequency interval v to v 2 at a frequency v Q , with radiation-free transmission (Förster transfer) to adjacent cross-linkers C and / or C "the photoactivatable end A and / or A 'of the cross-linker C and / or C" for reaction with the surface
  • F fluorescent ligand
  • FIG. 1 b B and F are linked via the flexible connection L. F can orient himself freely. There is no blanking as in FIG. 1a.
  • the lytic reagent specifically cut between monomers of the type R1 or R4 (circles) and monomers of the type of R3 or R6 (squares).
  • the cross-linker molecule C binds specifically to residues of the type of monomer R2 (triangles) and after photoactivation unspecifically to spatially adjacent groups, such as here to monomer R5.
  • FIG. 2b shows, instead of the fragment in FIG. 2a, two separate fragments which occur in the digestion of the macromolecule without a cross-linker.
  • the absorption frequency v Q of the fluorophores F must lie in a frequency interval in which there is no significant excitation of the photoactivatable groups.
  • the emission frequency of the fluorophores F must lie in a frequency interval v to v 2 in which a significant part of the photoactivatable groups A, A ', A "is activated on the cross-linker molecules.
  • the first step of the method according to the invention is preferably followed by a bioanalytical method step known per se in order to convert the result of the method according to the invention into information about the 3D structure of the macromolecule.
  • the main idea of the method according to the invention is that only those groups on the accessible surface of the macromolecule M are linked by cross-linker molecules C which are less distant from one another than the maximum length of the cross-linker molecule. If you can identify the groups linked in pairs by cross-linker molecules C, their maximum possible spatial distance is known, and information about the 3D structure has been obtained.
  • the process step known per se which is preferably connected to the first process step, comprises the following elements:
  • the cross-linking according to the invention is preferably combined with the direct photoactivation of cross-linkers C, as well as with the known method for cross-linking with bifunctional cross-linker molecules C.
  • the method according to the invention it is advisable to use the method according to the invention repeatedly in combination with the methods known per se which preferably follow it, as explained above.
  • Different parameters can be set in each cycle so that new, independent structural information is obtained.
  • different cross-linker molecules and digestive reagents can be selected in each cycle.
  • a fluorophore F specifically bound to the macromolecule M is the first important component of the method according to the invention.
  • the method according to the invention provides information about the 3D structure of the macromolecule M in the spatial environment of this fluorophore F. If, for example, the 3D structure in and around the substrate binding pocket of an enzyme is to be examined, fluorescent inhibitors come into consideration as carriers of the fluorophore F. or substrate analogs, as well as the substrate itself in the case of very slow-working enzymes, if it fluoresces.
  • fluorescent cofactors for example flavin in cholesterol oxidase
  • fluorescent effectors for example YC-1 as an effector of soluble guanylyl cyclase
  • fluorescent groups covalently linked to the macromolecule such as tryptophans in proteins. It is important in all cases that the position of the fluorophore F relative to the macromolecule M is determined as precisely as possible, which naturally results in the examples mentioned.
  • the fluorophore F is rigidly connected to part B of the molecule, which is responsible for the specific binding to the macromolecule M (Fig. 1a).
  • the fluorophore F is flexibly connected (for example via an alkyl chain) to part B, which is responsible for the specific binding to the macromolecule M. is ( Figure 1 b). Because of the flexible connection L, the fluorophore F can rotate freely relative to B and M.
  • both options provide different, complementary types of information about the 3D structure of the macromolecule.
  • the first step of the method according to the invention is the specific binding of the fluorophore-bearing ligand F to the macromolecule M, unless a fluorophore F already present in the macromolecule M is used.
  • the fluorescent ligand YC-1 can be used as a probe. YC-1 is placed in an aqueous solution of sGC and binds with high affinity in a specific binding pocket of sGC.
  • the second step of the method according to the invention is the chemically specific covalent bond between the non-photoactivatable end A and / or A 'of the cross linker (S in FIG. 1) on the one hand and suitable functional groups (m in FIG. 1) on the surface of the protein on the other hand .
  • the selected cross-linker C to the above solution in which the complexes between the macromolecule M and the fluorescent ligand F are already located.
  • C 4- [p-azido-salicylamidojbutylamine (ASBA) can be used as a cross-linker. With its amine end, ASBA reacts specifically with carbonyl and carboxyl groups on the surface of the macromolecule M.
  • the third step of the method according to the invention is the excitation of the fluorophore F by irradiation with light, the frequency of which corresponds to the absorption frequency of the fluorophore F.
  • the photo-activatable end A, A ', A "of the cross-linker C must not be directly activated at this frequency, which can generally be ensured by selecting suitable cross-linkers C.
  • the photo-activatable part of absorbs ASBA between 250 nm and 320 nm and can thus be excited with the emission wavelength of YC-1, which is in the same range.
  • the absorption and emission spectrum of the ligand F fluorescent in the binding pocket or of the fluorescent group can be shifted depending on the environment compared to the spectra of the free fluorophores F in aqueous or other solution (solvatochromism). This can be the case in particular (FIG. 1a) if the binding and the fluorescent part of the ligand F are rigidly connected.
  • the solvatochromic shift must be taken into account when selecting the cross-linker molecule C, since the fluorescent energy is to be transferred from the fluorophore F to the photo-activatable group of the cross-linker C, and therefore the photo-activatable group must absorb significantly at the emission frequency of the fluorophore F.
  • the absorption and emission frequencies of the fluorophore F are not known, they can be determined by fluorescence spectroscopy; the cross-linker C is then selected on the basis of the spectra measured in this way.
  • the cross-linker C can be used in parallel, which absorb at different frequency intervals, but not at the frequency of the light incident from outside (v Q ) to excite the fluorophore F. Is the solvatochromic shift of the fluorophore F depending on the Polarity of the surrounding medium is known, so it can be seen from the solvatochrome shift to the character that actually occurred of the binding pocket (polar or non-polar) are closed, whereby a first structural information about the binding pocket is already obtained.
  • the next step of the method according to the invention is the chemical activation of those photoactivatable groups A and / or A 'of the cross-linker molecules C and / or C "which are in spatial proximity to the fluorophore F.
  • the energy required for the activation is transferred via the forester -Transfer radiation-free transfer from the excited fluorophore F to the photoactivatable groups A and / or A '.
  • the transfer rate decreases in proportion to the sixth power of the reciprocal distance from F to A, A', A ".
  • the transmission rate depends not only on the distance between fluorophore F and photoactivatable group A, A ', A "but also on the spatial angle between the emission transition dipole moment of F and the absorption transition dipole moment of A, A', A". Highest rates are reached when both dipole moments are parallel, vanishing rates when they are orthogonal.
  • This effect is of importance in the process according to the invention when the fluorophore F is completely fixed relative to the macromolecule M (FIG. 1a). Then only those photoactivatable groups A are activated which are spatially adjacent to F and also have a suitable orientation relative to F. This effect generally does not occur since the photoactivatable groups usually have no preferred orientation before activation. Should the effect occur (Fig.
  • the radiation-free Förster transfer transfers the excitation energy from F to A and / or A 'much more efficiently than fluorescence emission from F and re-absorption by A and / or A'.
  • the latter process does not lead to a significant activation of A and / or A ⁇ , which simplifies the interpretation of the experiments.
  • the next step of the method according to the invention is the chemical reaction of the activated photoactivatable groups A and / or A 'of the crosslinker molecules C and / or C "with groups on the surface of the macromolecule M.
  • These reactions are chemically relatively unspecific, that is to say Activated groups A react with M to those groups that are immediately spatially adjacent to the activated groups A and / or A ', largely regardless of their chemical nature.
  • the cross-linker molecules C and / or C "form covalent bridges between Part of the surface of the macromolecule M.
  • the maximum length of these bridges can be influenced by the length and rigidity of the cross-linker C. At ASBA, this length is 1.6 nm.
  • the identification of the reacted groups of the macromolecule M thus provides further information about the 3D structure of the macromolecule M, namely an upper bound for the spatial distances between the fluorophore F and the groups on the surface of the macromolecule M that with the photoactivated groups A and / or A 'of the cross-linker molecules C and / or C "have reacted.
  • the method according to the invention is preferably used in combination with known bioanalytical and computational methods, which are explained in the following.
  • the macromolecule M is, for example, a protein
  • proteases such as trypsin can be used for this purpose. Trypsin cuts polypeptide chains specifically for lysines or arginines.
  • the same macromolecules M are digested in a further assay without cross-linkers C, C, C ". Mixtures of parts of the macromolecule M with or without attached cross-linkers C, C, C "are obtained from both digestion assays.
  • the mass spectrum of the digested macromolecule M without cross-linker C, C, C is compared with the mass spectrum of the digested macromolecule M with cross-linker C, C ⁇ C".
  • masses appear which correspond to the sum of the masses of the fragments linked via cross-linker C and / or C "and the mass of the cross-linker C.
  • These fragments are identified on the basis of the masses. It is thus known that these fragments are closer in the native structure of the macromolecule M than the maximum length of the cross-linker C.
  • the two linked parts and the space between them must be accessible to the cross-linker molecule C and / or C ". Both are important information about the 3D structure of the macromolecule.
  • the structural information obtained above is now being converted into an SD structural model.
  • the experimental results obtained by the method according to the invention are taken into account as geometrical boundary conditions in known methods for computer simulation (D. Hoffmann, E. W. Knapp; J. Phys. Chem. B 101: 6734-6740, 1997) of the macromolecule M.
  • these boundary conditions can also be used in distance-geometric methods and in threading methods for protein structure prediction.
  • the result of this computational step is an SD structural model or several 3D structural models of the macromolecule M.
  • the respective process steps can be carried out with other reagents.
  • cross linkers C different lengths or chemical specificity are used, as well as other digestive reagents are used. It is iterated until further refinement is no longer desired or possible.

Abstract

The invention relates to a method for the targeted chemical activation of photoactivatable cross-linking molecules around ligand binding pockets and fluorescent groups in macromolecules, especially biological macromolecules, by using fluorescent ligands or fluorescent groups of the macromolecule, and by selecting photoactivatable cross-linking molecules with specific activation energies, to achieve a radiation-free energy transfer (Forster transfer) from the fluorescent ligands or groups to the cross-linking molecules activated thereby. The invention also relates to a method for elucidating the 3-D structure of macromolecules (M), characterized in that a ligand (F) capable of fluorescence with a fluorescence frequency in the range (1 to (2 is introduced into the macromolecule (M) or its physical position to the macromolecule (M) is determined using known methods; one or more photoactivatable bifunctional cross-linking agents (C) with a corresponding excitation frequency in the range of (1 to (2) are bound in a covalent manner between the non-photoactivatable end (S) of the cross-linking agents (C, C', C") and suitable functional groups (m) of the macromolecule (M) in the absence of light; the macromolecule (M) is irradiated above the frequency interval (1 to (2 at a frequency (Q, and by means of a radiation-free transfer (Forster transfer) to neighbouring cross-linking agents (C and/or C"), the photoactivatable end (A and/or A') of the cross-linking element (C and/or C") is activated for reaction with the surface of the macromolecule (M) and reacted with the surface of said macromolecule (M) in accordance with the distance of the ligand (F) capable of fluorescence; the groups linked in pairs are identified using bioanalytical methods, especially specific digestion of the macromolecule (M), the digested fragments are divided, especially by mass, and physical proximities are determined by calculation.

Description

FLUORESZENZ-ENERGIE-TRANSFER VERMITTELTER CHEMISCHER AKTIVIERUNG (FETMA) FÜR DIE AUFKLÄRUNG DER 3D-STRUKTUR VON FLUORESCENCE ENERGY TRANSFER OF MEDIATED CHEMICAL ACTIVATION (FETMA) FOR THE ENLARGEMENT OF THE 3D STRUCTURE OF
BIOMAKROMOLEKÜLENBIOMACROMOLECULES
Die Erfindung betrifft ein Verfahren zur gezielten chemischen Aktivierung von photoaktivierbaren Cross-Linker-Molekülen um Ligand-Bindetaschen und fluoreszierende Gruppen in Makromolekülen, insbesondere biologischen Makromolekülen, durch Verwendung fluoreszierender Liganden des Makromoleküls und durch Wahl von photoaktivierbaren Cross-Linker-Molekülen mit spezifischen Aktivierungsenergien, so daß ein strahlungsloser Energietransfer (Förster- Transfer) von den fluoreszierenden Liganden zu den dadurch aktivierten Cross- Linker-Molekülen stattfindet.The invention relates to a method for the targeted chemical activation of photo-activatable cross-linker molecules around ligand binding pockets and fluorescent groups in macromolecules, in particular biological macromolecules, by using fluorescent ligands of the macromolecule and by selecting photo-activatable cross-linker molecules with specific activation energies. so that radiation-free energy transfer (Förster transfer) from the fluorescent ligands to the crosslinker molecules activated thereby takes place.
Das erfindungsgemäße Verfahren wird bevorzugt eingesetzt um ein an sich bekanntes bioanalytisches Verfahren zur Gewinnung von Information über die SD- Struktur von Biomakromolekülen auf funktioneil relevante Teile von Biomakromolekülen wie Ligand-Bindetaschen zu fokussieren. In diesem bioanalytischen Verfahren schließt sich an die Ausführung des erfindungsgemäßen Verfahrens an: ein spezifischer Verdau des Biomakromoleküls, eine Auftrennung der Bestandteile durch Chromatographie und Massenspek- trometrie, sowie eine Computersimulation zur Gewinnung von SD-Strukturmodellen mit der experimentell gewonnenen Information als Randbedingung. Zur Strukturverfeinerung kann das gesamte Verfahren in mehreren Iterationen durchlaufen werden.The method according to the invention is preferably used to focus a bioanalytical method known per se for obtaining information about the SD structure of biomacromolecules on functionally relevant parts of biomacromolecules such as ligand binding pockets. This bioanalytical method is followed by the execution of the method according to the invention: a specific digestion of the biomacromolecule, a separation of the components by chromatography and mass spectrometry, and a computer simulation to obtain SD structure models with the experimentally obtained information as a boundary condition. The entire process can be carried out in several iterations to refine the structure.
Biologische Makromoleküle wie Proteine, Ribonukleinsäuren oder makromolekulare Komplexe aus verschiedenen Biopolymeren wie zum Beispiel Ribosomen sind die Träger der essentiellen biochemischen Funktionen für fast alle Lebensprozesse in biologischen Organismen. Im allgemeinen sind diese Funktionen gebunden an die genau definierten 3D-Strukturen der Biomakromoleküle. Bei Kenntnis einer 3D-Struktur kann auf den funktioneilen Mechanismus geschlossen werden. Das macht 3D-Strukturen biologischer zu einer wichtigen Informationsquelle für molekulare Medizin und Pharmakologie. Besonders wertvoll sind Informationen über die 3D-Struktur der Bindetaschen biologischer Makromoleküle, da sie die eigentlich funktionell wichtigen Teile der Makromoleküle sind, weil dort Wechselwirkungen mit anderen gebundenen Molekülen wie Liganden oder Substraten stattfinden. Eines von vielen medizinisch relevanten Beispielen dafür, wie die Kenntnis der 3D-Struktur von Bindetaschen genutzt wird, ist das sogenannte rationale Design von Inhibitoren viraler oder bakterieller Enzyme. Diese Inhibitoren werden so entworfen, daß sie in die Bindetaschen der Enzyme passen wie Schlüssel in Schlösser. Dadurch werden die Enzyme blockiert und die Vermehrung der Viren oder Bakterien gestoppt. Ein solches Schlüssel-Schloß-Design ist nur dann gezielt möglich, wenn die SD- Struktur der Bindetasche bekannt ist.Biological macromolecules such as proteins, ribonucleic acids or macromolecular complexes from various biopolymers such as ribosomes are the carriers of essential biochemical functions for almost all life processes in biological organisms. In general, these functions are linked to the precisely defined 3D structures of the biomacromolecules. If a 3D structure is known, the functional mechanism can be concluded. This makes 3D biological structures an important source of information for molecular medicine and pharmacology. Information about the 3D structure of the binding pockets of biological macromolecules is particularly valuable because they are the actually functionally important parts of the macromolecules, because they interact with other bound molecules such as ligands or substrates. One of many medically relevant examples of how knowledge of the 3D structure of binding pockets is used is the so-called rational design of inhibitors of viral or bacterial enzymes. These inhibitors are designed to fit into the enzyme binding pockets like keys in locks. This blocks the enzymes and stops the virus or bacteria from multiplying. Such a key and lock design is only possible in a targeted manner if the SD structure of the binding pocket is known.
Im Stand der Technik ist die Bestimmung der 3D-Strukturen biologischer Makromoleküle mit Hilfe konventioneller Verfahren wie Röntgen- Kristallstrukturanalyse oder kernmagnetischer Resonanz im allgemeinen schwierig und zeitaufwendig. Die Gründe dafür sind vielfältig. Insbesondere werden in diesen Verfahren große (typischerweise millimolare) Mengen an aufgereinigtem Makromolekül in spezieller Form benötigt, entweder kristallin oder als konzentrierte Lösung. Darüber hinaus müssen zur Entschlüsselung der 3D- Struktur weitere schwierige Hürden überwunden werden, wie zum Beispiel in derIn the prior art, the determination of the 3D structures of biological macromolecules using conventional methods such as X-ray crystal structure analysis or nuclear magnetic resonance is generally difficult and time-consuming. The reasons for this are diverse. In particular, these processes require large (typically millimolar) amounts of purified macromolecule in a special form, either crystalline or as a concentrated solution. In addition, other difficult hurdles must be overcome to decode the 3D structure, such as in the
Kristallographie die Lösung des sogenannten Phasenproblems. Insgesamt kann so die Aufklärung einer 3D-Struktur eines Biomakromoleküls mehrereCrystallography the solution of the so-called phase problem. Overall, the elucidation of a 3D structure of a biomacromolecule can do several
Personenjahre beanspruchen.Claim person-years.
Es ist Aufgabe der Erfindung ein Verfahren zur 3D-Strukturaufklärung bereitzustellen, das geringe Mengen an reinem Makromolekül erfordert und auf funktionell relevante Bindetaschen fokussierbar ist. Darüber hinaus soll das erfindungsgemäße Verfahren in Fällen anwendbar sein, die besonders schwer mit konventionellen . Methoden zu behandeln sind. Dazu gehören beispielsweise Membranproteine sowie große, schwer zu reinigende und schwer zu kristallisierende globuläre Proteine und Komplexe. Das erfindungsgemäße Verfahren zur 3D-Strukturaufklärung von Makromolekülen (M) ist in einer ersten Ausführungsform dadurch gekennzeichnet, daß man einen fiuoreszenzfähigen Liganden (F) mit einer Fluoreszenzfrequenz im Bereich von v, bis v2 in das Makromolekül (M) einführt oder dessen räumliche Position zum Makromolekül (M) nach an sich bekannten Verfahren bestimmt, einen oder mehrere photoaktivierbare bifunktionelle Cross-Linker C, C\ C" mit einer jeweiligen Anregungsfrequenz im Bereich von v, bis v2 kovalent zwischen dem nicht-photoaktivierbaren Ende S des Cross-Linkers C, C\ C" und geeigneten funktioneilen Gruppen m des Makromoleküls M unter Lichtausschluß bindet, das Makromolekül M oberhalb des Frequenzintervalls v bis v2 mit einer Frequenz vQ bestrahlt, wobei mittels strahlungsloser Übertragung (Förster-Transfer) auf benachbarte Cross-Linker C und/oder C" das photoaktivierbare Ende A und/oder A' des Cross-Linkers C und/oder C" zur Reaktion mit der Oberfläche des Makromoleküls M aktiviert und mit der Oberfläche des Makromoleküls M in Abhängigkeit des Abstandes des fluoreszenzfähigen Liganden F reagiert und die paarweise verknüpften Gruppen durch bioanalytische Verfahren identifiziert, insbesondere spezifischen Verdau des Makromolküls M, die Verdaubruchstücke insbesondere nach Masse auftrennt und räumliche Nachbarschaften rechnerisch bestimmt.It is an object of the invention to provide a method for 3D structure elucidation, which requires small amounts of pure macromolecule and can be focused on functionally relevant binding pockets. In addition, the inventive method should be applicable in cases that are particularly difficult with conventional. Methods to be treated. These include, for example, membrane proteins as well as large, difficult to clean and difficult to crystallize globular proteins and complexes. In a first embodiment, the method according to the invention for 3D structure elucidation of macromolecules (M) is characterized in that a fluorescent ligand (F) with a fluorescence frequency in the range from v to v 2 is introduced into the macromolecule (M) or its spatial position determined to the macromolecule (M) by methods known per se, one or more photoactivatable bifunctional cross-linkers C, C \ C "with a respective excitation frequency in the range from v to v 2 covalently between the non-photoactivatable end S of the cross-linker C, C \ C "and suitable functional groups m of the macromolecule M binds with the exclusion of light, the macromolecule M is irradiated above the frequency interval v to v 2 at a frequency v Q , with radiation-free transmission (Förster transfer) to adjacent cross-linkers C and / or C "the photoactivatable end A and / or A 'of the cross-linker C and / or C" for reaction with the surface d It activates the macromolecule M and reacts with the surface of the macromolecule M as a function of the distance between the fluorescent ligand F and identifies the groups linked in pairs by bioanalytical methods, in particular specific digestion of the macromolecule M, which separates digest fragments, in particular by mass, and calculates spatial neighborhoods by calculation.
Weitere Ausführungsformen der Erfindung ergeben sich insbesondere aus den abhängigen Patentansprüchen.Further embodiments of the invention result in particular from the dependent patent claims.
Fig. 1a und Fig. 1 b beschreiben die chemische Aktivierung durch Förster- Transfer. Die Energie wird vom angeregten Fluorophor F auf benachbarte photoaktivierbare Gruppen A und/oder A' übertragen. Es findet keine nennenswerte Übertragung auf weiter entfernte photoaktivierbare Gruppen A" statt. M steht für Makromolekül; B steht für bindenden Teil des fluoreszierenden Liganden; F steht für Fluorophor; m steht für spezifische chemische Gruppen auf M; S steht für Gruppen auf Cross-Linker, die spezifisch kovalent an m-Gruppen binden; L steht für flexible Verbindung zwischen B und F, zum Beispiel eine Alkylkette. In Fig. 1 a sind B und F fest verbunden; es kann zum richtungsabhängigen Ausblenden von A-Gruppen kommen (A' wird im Gegensatz zu A nicht aktiviert, obwohl Abstand FA gleich dem Abstand FA' ist).1a and 1b describe the chemical activation by Förster transfer. The energy is transferred from the excited fluorophore F to neighboring photoactivatable groups A and / or A '. There is no significant transfer to more distant photoactivatable groups A ". M stands for macromolecule; B stands for the binding part of the fluorescent ligand; F stands for fluorophore; m stands for specific chemical groups on M; S stands for groups on cross-linkers that bind covalently to m groups; L stands for flexible connection between B and F, for example an alkyl chain. In Fig. 1 a B and F are firmly connected; Direction-dependent fading out of A groups can occur (in contrast to A, A 'is not activated, although distance FA is equal to distance FA').
In Fig. 1 b sind B und F über die flexible Verbindung L verknüpft. F kann sich frei orientieren. Es tritt keine Ausblendung wie in Fig. 1a auf.In Fig. 1 b, B and F are linked via the flexible connection L. F can orient himself freely. There is no blanking as in FIG. 1a.
Fig. 2 beschreibt die Zusammensetzung des Gemisches der makromolekularen Bruchstücke nach dem Verdau des Makromoleküls (schematisch). Das lytische Reagenz hat spezifisch geschnitten zwischen Monomeren des Typs von Monomer R1 oder R4 (Kreise) und Monomeren des Typs von Monomer R3 oder R6 (Quadrate). Das Cross-Linker Molekül C bindet spezifisch an Residuen vom Typ von Monomer R2 (Dreiecke) und nach Photoaktivierung unspezifisch an räumlich benachbarte Gruppen, wie hier an Monomer R5.2 describes the composition of the mixture of the macromolecular fragments after digestion of the macromolecule (schematic). The lytic reagent specifically cut between monomers of the type R1 or R4 (circles) and monomers of the type of R3 or R6 (squares). The cross-linker molecule C binds specifically to residues of the type of monomer R2 (triangles) and after photoactivation unspecifically to spatially adjacent groups, such as here to monomer R5.
Fig. 2a zeigt dieses Bruchstück als Bestandteil des Verdaus des Makromoleküls nach Cross-Linking.2a shows this fragment as part of the digestion of the macromolecule after cross-linking.
Fig. 2b zeigt statt des Bruchstücks in Fig. 2a zwei getrennte Bruchstücke, die im Verdau des Makromoleküls ohne Cross-Linker auftreten.2b shows, instead of the fragment in FIG. 2a, two separate fragments which occur in the digestion of the macromolecule without a cross-linker.
Das erfindungsgemäße Verfahren nutzt folgende physikalische Eigenschaften und Effekte:The method according to the invention uses the following physical properties and effects:
- Die spezifische räumlich definierte Bindung des fluoreszierenden Liganden (mit Fluorophor F in Fig. 1a und Fig. 1 b) an das Makromolekül. Statt eines Liganden kann auch eine natürlicherweise im Makromolekül vorhandene fluoreszierende Gruppe (zum Beispiel Indol-Gruppen von Tryptophan in Proteinen) genutzt werden, wenn die 3D-Struktur von dessen Umgebung untersucht werden soll.- The specific spatially defined binding of the fluorescent ligand (with fluorophore F in Fig. 1a and Fig. 1b) to the macromolecule. Instead of a ligand, a fluorescent group that is naturally present in the macromolecule (for example indole groups of tryptophan in proteins) can also be used if the 3D structure of its surroundings is to be examined.
- Die spezifische kovalente Bindung von Cross-Linker Molekül mit bestimmten zugänglichen Teilen des Makromoleküls über spezifische funktioneilen Gruppen (S in Fig. 1 a und Fig. 1 b) an einem Ende S der Cross-Linker Moleküle C, C, C". Am anderen Ende A, A', A" der Cross-Linker Moleküle befinden sich photoaktivierbare Gruppen (Akzeptoren A in Fig. 1a und Fig. 1 b).- The specific covalent binding of the cross-linker molecule with certain accessible parts of the macromolecule via specific functional groups (S in FIG. 1 a and FIG. 1 b) at one end S of the cross-linker molecules C, C, C ". At the other end A, A ', A "of the cross-linker molecules are photoactivatable groups (acceptors A in Fig. 1a and Fig. 1b).
- Die Anregung des Fluorophors des Liganden F oder geeignet modifizierter Liganden oder fluoreszierenden Gruppen.- The excitation of the fluorophore of ligand F or suitably modified ligands or fluorescent groups.
- Die strahlungslose, effiziente Übertragung (Förster-Transfer) der Fluoreszenzenergie von den gebundenen oder natürlicherweise im Makromolekül M vorhandenen Fluorophoren auf Akzeptoren A und/oder A' von Cross-Linker Molekülen C die zu den Fluorophoren räumlich benachbart sind.- The radiation-free, efficient transfer (Förster transfer) of the fluorescence energy from the fluorophores bound or naturally present in the macromolecule M to acceptors A and / or A 'of cross-linker molecules C which are spatially adjacent to the fluorophores.
- Die chemische Aktivierung der Akzeptoren A und/oder A' durch die Fluoreszenzenergie.- The chemical activation of acceptors A and / or A 'by the fluorescent energy.
- Die unspezifische kovalente Bindung der aktivierten Akzeptoren A und/oder A' an unmittelbar benachbarte Gruppen des Makromoleküls M (Cross-Linking).- The unspecific covalent binding of the activated acceptors A and / or A 'to directly adjacent groups of the macromolecule M (cross-linking).
Es ist für das erfindungsgemäße Verfahren von Bedeutung, daß folgende Bedingungen erfüllt sein sollten.It is important for the process according to the invention that the following conditions should be met.
- Die Absorptionsfrequenz vQ der Fluorophore F muß in einem Frequenzintervall liegen, in dem keine signifikante Anregung der photoaktivierbaren Gruppen stattfindet.- The absorption frequency v Q of the fluorophores F must lie in a frequency interval in which there is no significant excitation of the photoactivatable groups.
- Die Emissionsfrequenz der Fluorophore F muß in einem Frequenzintervall v, bis v2 liegen in dem ein signifikanter Teil der photoaktivierbaren Gruppen A, A', A" auf den Cross-Linker Molekülen aktiviert wird.- The emission frequency of the fluorophores F must lie in a frequency interval v to v 2 in which a significant part of the photoactivatable groups A, A ', A "is activated on the cross-linker molecules.
Diese beiden Bedingungen können durch geeignete Wahl des Liganden F und des Cross-Linker Moleküls C erreicht werden. Ein wesentlicher Unterschied des erfindungsgemäßen Verfahrens zu an sich bekannten Verfahren des Cross- Linkens mit photoaktivierbaren Molekülen, ist dessen indirekte Aktivierung über Anregung des Fluorophors, gefolgt von Förster-Transfer auf die photoaktivierbaren Gruppen A und/oder A'. Dadurch wird eine ortsaufgelöste Aktivierung rund um das spezifisch gebundene Fluorophor F möglich, mit anderen Worten eine Fokussierung der Aktivierung auf die Umgebung funktionell relevanter Bindetaschen oder Gruppen. Durch das erfindungsgemäße Verfahren wird also eine neue Qualität in der biologischen Strukturforschung erreicht.These two conditions can be achieved by a suitable choice of the ligand F and the cross-linker molecule C. A significant difference between the method according to the invention and known methods of crosslinking with photoactivatable molecules is its indirect activation via excitation of the fluorophore, followed by Förster transfer to the photoactivatable groups A and / or A '. This creates a spatially resolved Activation around the specifically bound fluorophore F possible, in other words focusing the activation on the environment of functionally relevant binding pockets or groups. The method according to the invention thus achieves a new quality in biological structure research.
An den ersten Schritt des erfindungsgemäßen Verfahrens schließt sich bevorzugt ein an sich bekannter bioanalytischer Verfahrensschritt an, um das Ergebnis des erfindungsgemäßen Verfahren in Information über die 3D-Struktur des Makromoleküls umzusetzen. Der Kerngedanke des erfindungsgemäßen Verfahrens ist, daß nur solche Gruppen auf der zugänglichen Oberfläche des Makromoleküls M durch Cross-Linker Moleküle C verknüpft werden, die eine geringere räumliche Entfernung voneinander haben, als die maximale Länge des Cross-Linker Moleküls. Kann man also die paarweise durch Cross-Linker Moleküle C verknüpften Gruppen identifizieren, so ist deren maximal mögliche räumliche Entfernung bekannt, und man hat eine Information über die 3D-Struktur gewonnen. Der an sich bekannte Verfahrensschritt, der bevorzugt an den ersten Verfahrensschritt angeschlossen wird, umfaßt folgende Elemente:The first step of the method according to the invention is preferably followed by a bioanalytical method step known per se in order to convert the result of the method according to the invention into information about the 3D structure of the macromolecule. The main idea of the method according to the invention is that only those groups on the accessible surface of the macromolecule M are linked by cross-linker molecules C which are less distant from one another than the maximum length of the cross-linker molecule. If you can identify the groups linked in pairs by cross-linker molecules C, their maximum possible spatial distance is known, and information about the 3D structure has been obtained. The process step known per se, which is preferably connected to the first process step, comprises the following elements:
- Spezifischer Verdau (Proteolyse, Nucleolyse) des Makromoleküls.- Specific digestion (proteolysis, nucleolysis) of the macromolecule.
- Auftrennung der Bruchstücke aus dem Verdau insbesondere nach Masse (HPLC, MALDI-TOF Massenspektrometrie).- Separation of the fragments from the digestion, especially by mass (HPLC, MALDI-TOF mass spectrometry).
- Rechnerische Bestimmung räumlicher Nachbarschaften aufgrund der experimentellen Resultate und Computersimulation des Makromoleküls mit den experimentell bestimmten Nachbarschaften als Randbedingungen. Aus diesem Schritt erhält man eines oder mehrere 3D-Strukturmodelle.- Computational determination of spatial neighborhoods based on the experimental results and computer simulation of the macromolecule with the experimentally determined neighborhoods as boundary conditions. One or more 3D structural models are obtained from this step.
Um die globale 3D-Struktur von Makromolekülen M zu bestimmen wird das erfindungsgemäße Cross-Linking bevorzugt kombiniert mit der direkten Photoaktiverung von Cross-Linkern C, sowie mit dem an sich bekannten Verfahren zum Cross-Linking mit bifunktionalen Cross-Linker Molekülen C. Zum Erreichen einer schrittweisen Verfeinerung des 3D-Strukturmodelles empfiehlt sich eine wiederholte Anwendung des erfindungsgemäßen Verfahrens in Kombination mit den bevorzugt daran sich anschließenden an sich bekannten Verfahren wie oben erläutert. In jedem Zyklus können unterschiedliche Parameter eingestellt werden, so daß neue unabhängige Strukturinformation gewonnen wird. Insbesondere können in jedem Zyklus andere Cross-Linker Moleküle und Verdauungs-Reagenzien gewählt werden.In order to determine the global 3D structure of macromolecules M, the cross-linking according to the invention is preferably combined with the direct photoactivation of cross-linkers C, as well as with the known method for cross-linking with bifunctional cross-linker molecules C. To achieve a step-by-step refinement of the 3D structure model, it is advisable to use the method according to the invention repeatedly in combination with the methods known per se which preferably follow it, as explained above. Different parameters can be set in each cycle so that new, independent structural information is obtained. In particular, different cross-linker molecules and digestive reagents can be selected in each cycle.
1. Spezifische räumlich definierte Bindung von Ligand an Makromolekül1. Specific spatially defined binding of ligand to macromolecule
Ein spezifisch an das Makromolekül M gebundenes Fluorophor F ist die erste wichtige Komponente des erfindungsgemäßen Verfahrens. Das erfindungsgemäße Verfahren liefert Information über die 3D-Struktur des Makromoleküls M in der räumlichen Umgebung dieses Fluorophors F. Soll zum Beispiel die 3D-Struktur in und um die Substrat-Bindetasche eines Enzyms untersucht werden, kommen als Träger des Fluorophors F in Betracht fluoreszierende Inhibitoren oder Substratanaloge, sowie bei sehr langsam arbeitenden Enzymen auch das Substrat selbst, falls es fluoresziert. Daneben können fluoreszierende Kofaktoren (zum Beispiel Flavin in Cholesterol Oxidase) oder fluoreszierende Effektoren genutzt werden (zum Beispiel YC-1 als Effektor der löslichen Guanylylcyclase), sowie kovalent mit dem Makromolekül verbundene fluoreszierende Gruppen wie Tryptophane in Proteinen. Wichtig ist in allen Fällen, daß die Position des Fluorophors F relativ zum Makromolekül M möglichst genau bestimmt ist, was sich in den genannten Beispielen natürlicherweise ergibt.A fluorophore F specifically bound to the macromolecule M is the first important component of the method according to the invention. The method according to the invention provides information about the 3D structure of the macromolecule M in the spatial environment of this fluorophore F. If, for example, the 3D structure in and around the substrate binding pocket of an enzyme is to be examined, fluorescent inhibitors come into consideration as carriers of the fluorophore F. or substrate analogs, as well as the substrate itself in the case of very slow-working enzymes, if it fluoresces. In addition, fluorescent cofactors (for example flavin in cholesterol oxidase) or fluorescent effectors (for example YC-1 as an effector of soluble guanylyl cyclase) can be used, as well as fluorescent groups covalently linked to the macromolecule such as tryptophans in proteins. It is important in all cases that the position of the fluorophore F relative to the macromolecule M is determined as precisely as possible, which naturally results in the examples mentioned.
Zu unterscheiden im Sinne des erfindungsgemäßen Verfahrens sind zwei Arten der Verknüpfung des Fluorophors mit dem der Rest der Gruppe die spezifisch an das Makromolekül gebunden ist (Fig. 1 ):In the sense of the method according to the invention, a distinction must be made between two types of linkage of the fluorophore with which the rest of the group is specifically bound to the macromolecule (FIG. 1):
- Das Fluorophor F ist starr mit dem Teil B des Moleküls verbunden, der für die spezifische Bindung an das Makromolekül M verantwortlich ist (Fig. 1a).- The fluorophore F is rigidly connected to part B of the molecule, which is responsible for the specific binding to the macromolecule M (Fig. 1a).
- Das Fluorophor F ist flexibel (zum Beispiel über eine Alkylkette) mit dem Teil B verbunden, der für die spezifische Bindung an das Makromolekül M verantwortlich ist (Figur 1 b). Wegen der flexiblen Verbindung L kann das Fluorophor F frei rotieren relativ zu B und M.- The fluorophore F is flexibly connected (for example via an alkyl chain) to part B, which is responsible for the specific binding to the macromolecule M. is (Figure 1 b). Because of the flexible connection L, the fluorophore F can rotate freely relative to B and M.
Wie unten in Punkt 4 ausgeführt liefern beide Möglichkeiten unterschiedliche, sich ergänzende Arten von Information über die 3D-Struktur des Makromoleküls.As explained in point 4 below, both options provide different, complementary types of information about the 3D structure of the macromolecule.
Zusammenfassend ist also der erste Schritt des erfindungsgemäßen Verfahrens die spezifische Bindung des Fluorophor-tragenden Liganden F an das Makromolekül M, falls nicht ein bereits im Makromolekül M vorhandenes Fluorophor F genutzt wird. Soll zum Beispiel die Umgebung der Effektorbindetasche der löslichen Guanylylcyclase (sGC) untersucht werden, so kann der fluoreszierende Ligand YC-1 als Sonde verwendet werden. YC-1 wird in eine wäßrige Lösung von sGC gegeben und bindet mit hoher Affinität in einer spezifischen Bindetasche von sGC.In summary, the first step of the method according to the invention is the specific binding of the fluorophore-bearing ligand F to the macromolecule M, unless a fluorophore F already present in the macromolecule M is used. For example, if the area around the effector binding pocket of soluble guanylyl cyclase (sGC) is to be examined, the fluorescent ligand YC-1 can be used as a probe. YC-1 is placed in an aqueous solution of sGC and binds with high affinity in a specific binding pocket of sGC.
2. Spezifische chemische Bindung der Cross-Linkers C2. Specific chemical binding of the cross-linkers C
Der zweite Schritt des erfindungsgemäßen Verfahrens ist die chemischspezifische kovalente Bindung zwischen dem nicht-photoaktivierbaren Ende A und/oder A' des Cross-Linkers (S in Figur 1 ) einerseits und passenden funktioneilen Gruppen (m in Figur 1 ) auf der Oberfläche des Proteins andererseits. Dazu gibt man den ausgewählten Cross-Linker C in die obige Lösung in der sich bereits die Komplexe zwischen dem Makromolekül M und dem fluoreszierenden Liganden F befinden. Zum Beispiel kann als Cross-Linker C 4-[p-Azido- salicylamidojbutylamin (ASBA) verwendet werden. ASBA reagiert mit seinem Amin-Ende spezifisch mit Carbonyl- und Carboxyl-Gruppen auf der Oberfläche des Makromoleküls M. Es ist wichtig die Reaktion in der Dunkelheit stattfinden zu lassen um eine vorzeitige Photoaktivierung von ASBA zu verhindern. Außerdem muß die Lösung so eingestellt werden, daß es zwar zu einer Reaktion von ASBA mit dem Makromolekül M kommt, nicht aber zu einer Denaturierung des Makromoleküls M. 3. Anregung des Fluorophors FThe second step of the method according to the invention is the chemically specific covalent bond between the non-photoactivatable end A and / or A 'of the cross linker (S in FIG. 1) on the one hand and suitable functional groups (m in FIG. 1) on the surface of the protein on the other hand . To do this, add the selected cross-linker C to the above solution in which the complexes between the macromolecule M and the fluorescent ligand F are already located. For example, C 4- [p-azido-salicylamidojbutylamine (ASBA) can be used as a cross-linker. With its amine end, ASBA reacts specifically with carbonyl and carboxyl groups on the surface of the macromolecule M. It is important to allow the reaction to take place in the dark to prevent premature photoactivation of ASBA. In addition, the solution must be adjusted so that there is a reaction of ASBA with the macromolecule M, but not a denaturation of the macromolecule M. 3. Excitation of the fluorophore F
Der dritte Schritt des erfindungsgemäßen Verfahrens ist die Anregung des Fluorophors F durch Einstrahlung von Licht, dessen Frequenz der Absorptionsfrequenz des Fluorophors F entspricht. Wie bereits oben beschrieben, darf bei dieser Frequenz keine direkt Aktivierung des photoaktivierbaren Endes A, A', A" des Cross-Linkers C erfolgen, was im allgemeinen durch Wahl geeigneter Cross- Linker C gewährleistet werden kann. Zum Beispiel absorbiert der photoaktivierbare Teil von ASBA zwischen 250 nm und 320 nm und läßt sich damit bei der Emissionsweilenlänge von YC-1 anregen, die im selben Bereich liegt.The third step of the method according to the invention is the excitation of the fluorophore F by irradiation with light, the frequency of which corresponds to the absorption frequency of the fluorophore F. As already described above, the photo-activatable end A, A ', A "of the cross-linker C must not be directly activated at this frequency, which can generally be ensured by selecting suitable cross-linkers C. For example, the photo-activatable part of absorbs ASBA between 250 nm and 320 nm and can thus be excited with the emission wavelength of YC-1, which is in the same range.
Es ist zu beachten, daß Absorbtions- und Emissions-Spektrum des in der Bindetasche fluoreszierenden Liganden F oder der fluoreszierenden Gruppe abhängig von der Umgebung verschoben sein kann gegenüber den Spektren der freien Fluorophore F in wäßriger oder anderer Lösung (Solvatochromie). Das kann insbesondere dann der Fall sein (Figur 1a), wenn der bindende und der fluoreszierende Teil des Liganden F starr verbunden sind. Die solvatochrome Verschiebung muß bei der Auswahl des Cross-Linker Moleküls C berücksichtigt werden, da die Fluoreszenzenergie vom Fluorophor F auf die photoaktivierbare Gruppe des Cross-Linkers C übertragen werden soll, und daher die photoaktiverbare Gruppe bei der Emissionsfrequenz des Fluorophors F signifikant absorbieren muß. Sind die Absorptions- und Emissionsfrequenzen des Fluorophors F nicht bekannt, können sie fluoreszenzspektroskopisch bestimmt werden; anhand der so gemessenen Spektren wird dann der Cross-Linker C ausgewählt. Alternativ können parallel mehrere Typen von Cross-Linkern C verwendet werden, die in unterschiedlichen Frequenzintervallen absorbieren, nicht aber bei der Frequenz des von außen eingestrahlten Lichtes (vQ) zur Anregung des Fluorophors F. Ist die solvatochrome Verschiebung des Fluorophors F in Abhängigkeit von der Polarität des umgebenden Mediums bekannt, so kann aus der tatsächlich aufgetretenen solvatochromen Verschiebung auf den Charakter der Bindungstasche (polar oder nicht-polar) geschlossen werden, womit bereits eine erste Strukturinformation über die Bindetasche gewonnen ist.It should be noted that the absorption and emission spectrum of the ligand F fluorescent in the binding pocket or of the fluorescent group can be shifted depending on the environment compared to the spectra of the free fluorophores F in aqueous or other solution (solvatochromism). This can be the case in particular (FIG. 1a) if the binding and the fluorescent part of the ligand F are rigidly connected. The solvatochromic shift must be taken into account when selecting the cross-linker molecule C, since the fluorescent energy is to be transferred from the fluorophore F to the photo-activatable group of the cross-linker C, and therefore the photo-activatable group must absorb significantly at the emission frequency of the fluorophore F. If the absorption and emission frequencies of the fluorophore F are not known, they can be determined by fluorescence spectroscopy; the cross-linker C is then selected on the basis of the spectra measured in this way. Alternatively, several types of cross-linkers C can be used in parallel, which absorb at different frequency intervals, but not at the frequency of the light incident from outside (v Q ) to excite the fluorophore F. Is the solvatochromic shift of the fluorophore F depending on the Polarity of the surrounding medium is known, so it can be seen from the solvatochrome shift to the character that actually occurred of the binding pocket (polar or non-polar) are closed, whereby a first structural information about the binding pocket is already obtained.
4. Chemische Aktivierung durch strahlungslose Übertragung der Fluoreszenzenergie auf räumlich benachbarte spezifische Akzeptoren4. Chemical activation by radiationless transfer of the fluorescence energy to spatially neighboring specific acceptors
Der nächste Schritt des erfindungsgemäßen Verfahrens ist die chemische Aktivierung jener photoaktivierbaren Gruppen A und/oder A' der Cross-Linker Moleküle C und/oder C", die sich in räumlicher Nähe zum Fluorophor F befinden. Die zur Aktivierung notwendige Energie wird über den Förster-Transfer strahlungsios vom angeregten Fluorophor F auf die photoaktivierbaren Gruppen A und/oder A' übertragen. Die Übertragungsrate sinkt proportional zur sechsten Potenz des reziproken Abstandes von F zu A, A', A" ab. Hohe Übertragungsraten gibt es typischerweise wenn die Entfernung zwischen F und A, A', A" in der Größenordnung von 1 nm liegt. Zu größeren Entfernungen hin nimmt die Übertragungsrate rapide ab.The next step of the method according to the invention is the chemical activation of those photoactivatable groups A and / or A 'of the cross-linker molecules C and / or C "which are in spatial proximity to the fluorophore F. The energy required for the activation is transferred via the forester -Transfer radiation-free transfer from the excited fluorophore F to the photoactivatable groups A and / or A '. The transfer rate decreases in proportion to the sixth power of the reciprocal distance from F to A, A', A ". There are typically high transmission rates if the distance between F and A, A ', A "is of the order of 1 nm. The transmission rate decreases rapidly as the distance increases.
Die Übertragungsrate hängt nicht nur ab von der Entfernung zwischen Fluorophor F und photoaktivierbarer Gruppe A, A', A" sondern auch vom räumlichen Winkel zwischen dem Emissions-Übergangsdipolmoment von F und dem Absorbtions- Übergangsdipolmoment von A, A', A". Höchste Raten werden erreicht, wenn beide Dipolmomente parallel sind, verschwindende Raten wenn sie orthogonal stehen. Dieser Effekt ist im erfindungsgemäßen Verfahren dann von Bedeutung, wenn das Fluorophor F ist relativ zum Makromolekül M völlig fixiert (Fig. 1a). Dann werden nur solche photoaktivierbaren Gruppen A aktiviert, die sowohl räumlich benachbart zu F sind, als auch eine geeignete Orientierung relativ zu F aufweisen. Dieser Effekt tritt im allgemeinen nicht auf, da die photoaktivierbaren Gruppen vor der Aktivierung meist keine Vorzugsorientierung aufweisen. Sollte der Effekt doch auftreten (Fig. 1 a, nur A wird aktiviert, A' dagegen nicht, obwohl beide Gruppen gleiche Entfernung zu F haben), so empfiehlt sich die Verwendung eines Liganden mit flexibler Verbindung L zwischen dem bindenden Teil des Liganden B und dem Fluorophor F (Fig. 1 b). Da F dann frei orientieren kann, wird eine Ausblendung photoaktivierbarer Gruppen aufgrund der Orientierungsabhängigkeit vermieden, das heißt allein der Abstand zwischen F und A bestimmt die Aktivierung.The transmission rate depends not only on the distance between fluorophore F and photoactivatable group A, A ', A "but also on the spatial angle between the emission transition dipole moment of F and the absorption transition dipole moment of A, A', A". Highest rates are reached when both dipole moments are parallel, vanishing rates when they are orthogonal. This effect is of importance in the process according to the invention when the fluorophore F is completely fixed relative to the macromolecule M (FIG. 1a). Then only those photoactivatable groups A are activated which are spatially adjacent to F and also have a suitable orientation relative to F. This effect generally does not occur since the photoactivatable groups usually have no preferred orientation before activation. Should the effect occur (Fig. 1 a, only A is activated, A 'not, although both groups are at the same distance from F), it is advisable to use a ligand with a flexible connection L between the binding part of ligand B and the fluorophore F (Fig. 1 b). Since F can then freely orient itself, a masking of photoactivatable groups becomes due to the orientation dependence avoided, that means only the distance between F and A determines the activation.
Der strahlungslose Förster-Transfer überträgt die Anregungsenergie von F sehr viel effizienter auf A und/oder A' als Fluoreszenz-Emission von F und ReAbsorption durch A und/oder A'. Damit führt der letztere Prozeß nicht zu einer signifikanten Aktivierung von A und/oder A\ was die Deutung der Experimente vereinfacht.The radiation-free Förster transfer transfers the excitation energy from F to A and / or A 'much more efficiently than fluorescence emission from F and re-absorption by A and / or A'. Thus the latter process does not lead to a significant activation of A and / or A \, which simplifies the interpretation of the experiments.
5. Chemisch unspezifische Reaktion der photoaktivierbaren Gruppen der Cross- Linker Moleküle C und/oder C"5. Chemically non-specific reaction of the photoactivatable groups of the cross-linker molecules C and / or C "
Der nächste Schritt des erfindungsgemäßen Verfahrens ist die chemische Reaktion der aktivierten photoaktivierbaren Gruppen A und/oder A' der Cross- Linker Molküle C und/oder C" mit Gruppen auf der Oberfläche des Makromoleküls M. Diese Reaktionen sind chemisch relativ unspezifisch, das heißt die aktivierten Gruppen A reagieren mit solchen Gruppen auf M, die unmittelbar räumlich benachbart zu den aktivierten Gruppen A und/oder A' sind, weitgehend ungeachtet deren chemischer Natur. Nach der Reaktion bilden die Cross-Linker Moleküle C und/oder C" kovalente Brücken zwischen Teilen der Oberfläche des Makromoleküls M. Die maximale Länge dieser Brücken läßt sich durch die Länge und Steifigkeit des Cross-Linkers C beeinflussen. Bei ASBA liegt diese Länge bei 1.6 nm.The next step of the method according to the invention is the chemical reaction of the activated photoactivatable groups A and / or A 'of the crosslinker molecules C and / or C "with groups on the surface of the macromolecule M. These reactions are chemically relatively unspecific, that is to say Activated groups A react with M to those groups that are immediately spatially adjacent to the activated groups A and / or A ', largely regardless of their chemical nature. After the reaction, the cross-linker molecules C and / or C "form covalent bridges between Part of the surface of the macromolecule M. The maximum length of these bridges can be influenced by the length and rigidity of the cross-linker C. At ASBA, this length is 1.6 nm.
Im allgemeinen werden nur solche Cross-Linker C mit dem Makromolekül M reaktiv, deren Abstand zum Fluorophor F gering ist. Die Identifizierung der reagierten Gruppen des Makromoleküls M liefert damit eine weitere Information über die 3D-Struktur des Makromoleküls M, nämlich eine obere Schranke für die räumliche Entfernungen zwischen dem Fluorophor F und den Gruppen auf der Oberfläche des Makromoleküls M die mit den photoaktivierten Gruppen A und/oder A' der Cross-Linker Moleküle C und/oder C" reagiert haben. Um die Resultate der Anwendung des erfindungsgemäßen Verfahrens in SD- Strukturinformation umzusetzen, wird das erfindungsgemäße Verfahren bevorzugt in Kombination mit an sich bekannten bioanalytischen und rechnerischen Verfahren eingesetzt, die im folgenden erläutert werden.In general, only those cross-linkers C become reactive with the macromolecule M whose distance from the fluorophore F is small. The identification of the reacted groups of the macromolecule M thus provides further information about the 3D structure of the macromolecule M, namely an upper bound for the spatial distances between the fluorophore F and the groups on the surface of the macromolecule M that with the photoactivated groups A and / or A 'of the cross-linker molecules C and / or C "have reacted. In order to convert the results of the application of the method according to the invention into SD structure information, the method according to the invention is preferably used in combination with known bioanalytical and computational methods, which are explained in the following.
6. Spezifischer Verdau6. Specific digestion
Die Makromoleküle M mit assoziierten Fluorophoren F und Cross-Linkern C, C, C" werden spezifisch verdaut, das heißt mit lytischen Reagenzien zerschnitten, die an spezifischen Motiven auf den Makromolekülen M reagieren. Handelt es sich bei dem Makromolekül M zum Beispiel um ein Protein, so kann man zu diesen Zweck Proteasen wie Trypsin einsetzen. Trypsin schneidet Polypeptid- ketten spezifisch nach Lysinen oder Argininen. Zu Vergieichszwecken werden in einem weiteren Assay die gleichen Makromoleküle M ohne Cross-Linker C, C, C" verdaut. Aus beiden Verdauungs-Assays erhält man jeweils Gemische von Teilen des Makromoleküls M mit beziehungsweise ohne angeheftete Cross-Linker C, C, C". Wichtig sind solche Teile, die im Makromolekül M ohne Cross-Linker C, C, C" nach dem Verdau in zwei getrennte Bruchstücke zerfallen (Fig. 2b), während sie nach Anwendung des erfindungsgemäßen Verfahrens durch Cross-Linker C und/oder C" zusammengehalten werden (Fig. 2a). Solche Teile haben in der nativen Konformation des Makromoleküls M einen räumlichen Abstand, der durch die Länge des Cross-Linker Moleküls C gegeben ist.The macromolecules M with associated fluorophores F and cross-linkers C, C, C "are digested specifically, that is cut up with lytic reagents which react on specific motifs on the macromolecules M. If the macromolecule M is, for example, a protein , proteases such as trypsin can be used for this purpose. Trypsin cuts polypeptide chains specifically for lysines or arginines. For comparison purposes, the same macromolecules M are digested in a further assay without cross-linkers C, C, C ". Mixtures of parts of the macromolecule M with or without attached cross-linkers C, C, C "are obtained from both digestion assays. Those parts that are in the macromolecule M without cross-linkers C, C, C" after digestion are important break up into two separate fragments (FIG. 2b) while they are held together by cross-linkers C and / or C "(FIG. 2a) after the method according to the invention has been applied. Such parts have a spatial spacing in the native conformation of the macromolecule M. which is given by the length of the cross-linker molecule C.
7. Trennung und Identifizierung der makromolekularen Bruchstücke7. Separation and identification of the macromolecular fragments
Zum Aufbau eines 3D-Strukturmodelles wird noch die Information benötigt, welche Teile des Makromoleküls M durch Cross-Linker C verknüpft wurden. Die Gemische der obigen Bruchstücke müssen also aufgetrennt und die einzelnen Teile identifiziert werden, das heißt Sequenzstücken des makromolekularen Polymers zugeordnet werden (die Kenntnis der Sequenz des Polymers, zum Beispiel im Falle eines Proteins die Sequenz der Aminosäuren, ist hier vorausgesetzt). Zur Auftrennung und Identifizierung werden im erfindungsgemäßen Verfahren bevorzugt an sich bekannten Verfahren wie HPLC und MALDI-TOF Massenspektrometrie eingesetzt. Anhand der Massen der Bruchstücke können diese im allgemeinen identifiziert werden.To build a 3D structural model, information is still required as to which parts of the macromolecule M have been linked by cross-linker C. The mixtures of the above fragments must therefore be separated and the individual parts identified, i.e. assigned to sequence pieces of the macromolecular polymer (knowledge of the sequence of the polymer, for example in the case of a protein the sequence of the amino acids is required here). For separation and identification, methods known per se, such as HPLC and MALDI-TOF mass spectrometry used. The masses of the fragments can generally be used to identify them.
Das Massenspektrum des verdauten Makromoleküls M ohne Cross-Linker C, C, C" wird verglichen mit dem Massenspektrum des verdauten Makromoleküls M mit Cross-Linker C, C\ C". In letzterem tauchen Massen auf, die der Summe der Massen der über Cross-Linker C und/oder C" verknüpften Bruchstücke und der Masse des Cross-Linkers C entsprechen. Anhand der Massen werden diese Bruchstücke identifiziert. Es ist damit bekannt, daß diese Bruchstücke sich in der nativen Struktur des Makromoleküls M näher sind als die maximale Länge des Cross-Linkers C. Außerdem ist damit bekannt, daß die beiden verknüpften Teile und der Raum dazwischen dem Cross-Linker Molekül C und/oder C" zugänglich sein muß. Beides sind wichtige Informationen zur 3D-Struktur des Makromoleküls.The mass spectrum of the digested macromolecule M without cross-linker C, C, C "is compared with the mass spectrum of the digested macromolecule M with cross-linker C, C \ C". In the latter, masses appear which correspond to the sum of the masses of the fragments linked via cross-linker C and / or C "and the mass of the cross-linker C. These fragments are identified on the basis of the masses. It is thus known that these fragments are closer in the native structure of the macromolecule M than the maximum length of the cross-linker C. In addition, it is known that the two linked parts and the space between them must be accessible to the cross-linker molecule C and / or C ". Both are important information about the 3D structure of the macromolecule.
8. Rechnerische Vorhersage der 3D-Struktur des Makromoleküls mit den experimentell bestimmten Informationen als Randbedingungen8. Computer prediction of the 3D structure of the macromolecule with the experimentally determined information as boundary conditions
Die oben gewonnene Strukturinformation wird nun noch umgesetzt in ein SD- Strukturmodell. Dazu werden die durch das erfindungsgemäße Verfahren gewonnenen experimentellen Resultate als geometrische Randbedingungen in an sich bekannten Verfahren zur Computersimulation (D. Hoffmann, E. W. Knapp; J. Phys. Chem. B 101 :6734-6740, 1997) des Makromoleküls M berücksichtigt. Alternativ können diese Randbedinungen auch in distanzgeometrischen Verfahren, sowie in Threading-Verfahren zur Proteinstrukturvorhersage eingesetzt werden. Resultat dieses rechnerischen Schrittes ist in jedem Fall ein SD- Strukturmodell oder mehrere 3D-Strukturmodelle des Makromoleküls M.The structural information obtained above is now being converted into an SD structural model. For this purpose, the experimental results obtained by the method according to the invention are taken into account as geometrical boundary conditions in known methods for computer simulation (D. Hoffmann, E. W. Knapp; J. Phys. Chem. B 101: 6734-6740, 1997) of the macromolecule M. Alternatively, these boundary conditions can also be used in distance-geometric methods and in threading methods for protein structure prediction. In any case, the result of this computational step is an SD structural model or several 3D structural models of the macromolecule M.
9. Weitere Durchläufe mit anderen Cross-Linkern und lyrischen Reagenzien9. Further runs with other cross-linkers and lyrical reagents
Falls eine Verfeinerung des 3D-Strukturmodells gewünscht ist, oder die Anzahl der mit den experimentellen Randbedingungen vereinbaren 3D-Strukturmodelle verringert werden soll, so können die jeweiligen Verfahrensschritte mit anderen Reagenzien durchlaufen werden. Insbesondere können Cross-Linker C verschiedener Länge oder chemischer Spezifität verwendet werden, sowie andere Verdauungsreagenzien eingesetzt werden. Es wird solange iteriert, bis eine weitere Verfeinerung nicht mehr gewünscht oder möglich ist. If a refinement of the 3D structure model is desired, or if the number of 3D structure models compatible with the experimental boundary conditions is to be reduced, the respective process steps can be carried out with other reagents. In particular, cross linkers C different lengths or chemical specificity are used, as well as other digestive reagents are used. It is iterated until further refinement is no longer desired or possible.

Claims

PATENTANSPRÜCHE PATENT CLAIMS
1. Verfahren zur 3D-Strukturaufklärung von Makromolekülen (M), dadurch gekennzeichnet, daß man einen fluoreszenzfähigen Liganden (F) mit einer Fluoreszenzfrequenz im Bereich von v1 bis v2 in das Makromolekül (M) einführt oder dessen räumliche Position zum Makromolekül (M) nach an sich bekannten Verfahren bestimmt, einen oder mehrere photoaktivierbare bifunktionelle Cross-Linker (C) mit einer jeweiligen Anregungsfrequenz im Bereich von v., bis v2 kovalent zwischen dem nicht-photoaktivierbaren Ende (S) des Cross-Linkers (C, C, C") und geeigneten funktionellen Gruppen (m) des Makromoleküls (M) unter Lichtausschluß bindet, das Makromolekül (M) oberhalb des Frequenzintervalls v., bis v2 mit einer Frequenz vQ bestrahlt, wobei mittels strahlungsloser Übertragung (Förster- Transfer) auf benachbarte Cross-Linker (C und/oder C") das photoaktivierbare Ende (A und/oder A') des Cross-Linkers (C und/oder C") zur Reaktion mit der Oberfläche des Makromoleküls (M) aktiviert und mit der Oberfläche des Makromoleküls (M) in Abhängigkeit des Abstandes des fluoreszenzfähigen Liganden (F) reagiert und die paarweise verknüpften Gruppen durch bioanalytische Verfahren identifiziert, insbesondere spezifischen Verdau des Makromolküls (M), die Verdaubruchstücke insbesondere nach Masse auftrennt und räumliche Nachbarschaften rechnerisch bestimmt.1. A process for the 3D structure elucidation of macromolecules (M), characterized in that one introduces a fluorescent ligand (F) with a fluorescence frequency in the range from v 1 to v 2 into the macromolecule (M) or its spatial position to the macromolecule (M ) determined by methods known per se, one or more photoactivatable bifunctional cross-linkers (C) with a respective excitation frequency in the range from v. to v 2 covalently between the non-photoactivatable end (S) of the cross-linker (C, C , C ") and suitable functional groups (m) of the macromolecule (M) binds with the exclusion of light, the macromolecule (M) above the frequency interval v., To v 2 is irradiated with a frequency v Q , with radiation-free transmission (Förster transfer) on adjacent cross-linkers (C and / or C ") the photoactivatable end (A and / or A ') of the cross-linker (C and / or C") for reaction with the surface of the macromolecule (M) is activated and with the surface of the macromolecule (M) reacts depending on the distance of the fluorescent ligand (F) and identifies the groups linked in pairs by bioanalytical methods, in particular specific digestion of the macromolecule (M), which separates digest fragments, in particular by mass, and calculates spatial neighborhoods by calculation.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß man die Makromoleküle (M) auswählt aus Proteinen, Ribonukleinsäuren oder makromolekularen Komplexen verschiedener Biopolymeren.2. The method according to claim 1, characterized in that the macromolecules (M) are selected from proteins, ribonucleic acids or macromolecular complexes of different biopolymers.
3. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß man gleichzeitig die direkte Photoaktivierung von Cross-Linkern (C) sowie auch bifunktionelle chemisch spezifisch bindende Cross-Linker einsetzt. 3. The method according to claim 1, characterized in that simultaneously the direct photoactivation of cross-linkers (C) and also bifunctional chemically specific binding cross-linkers are used.
4. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß man als Cross- Linker 4-[p-Azidosalicylamido]butylamin (ASBA) einsetzt.4. The method according to claim 1, characterized in that 4- [p-azidosalicylamido] butylamine (ASBA) is used as crosslinker.
5. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß man die Verdaubruchstücke nach Masse auftrennt.5. The method according to claim 1, characterized in that the digest pieces are separated by mass.
6. Verfahren nach einem der Ansprüche 1 bis 5, , dadurch gekennzeichnet, daß man das Verfahren mehrfach wiederholt, gegebenenfalls verschiedene fluores- zenzfähige Liganden (F), Cross-Linker (C) und /oder Verdauungs-Reagenzien einsetzt. 6. The method according to any one of claims 1 to 5, characterized in that the process is repeated several times, optionally using different fluorescent ligands (F), cross-linker (C) and / or digestive reagents.
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