EP1057020A2 - Activation chimique induite par transfert d'energie de fluorescence (fetma) pour l'exploration de la structure tridimensionnelle de biomacromolecules - Google Patents

Activation chimique induite par transfert d'energie de fluorescence (fetma) pour l'exploration de la structure tridimensionnelle de biomacromolecules

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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
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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Withdrawn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)
English (en)
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 Foerderung der Angewandten Forschung eV
Original Assignee
GMD Forschungszentrum Informationstechnik GmbH
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Publication date
Application filed by GMD Forschungszentrum Informationstechnik GmbH filed Critical GMD Forschungszentrum Informationstechnik GmbH
Publication of EP1057020A2 publication Critical patent/EP1057020A2/fr
Withdrawn legal-status Critical Current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.

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Abstract

L'invention concerne un procédé d'activation chimique ciblée de molécules d'agent réticulant photoactivables au niveau de poches de liaison de ligands et de groupes fluorescents se trouvant dans les macromolécules, en particulier des macromolécules biologiques, au moyen de ligands fluorescents ou de groupes fluorescents desdites macromolécules et par sélection de molécules d'agent réticulant photoactivables présentant une énergie d'activation spécifique, un transfert d'énergie sans rayonnement (transfert de Förster) se faisant des ligands ou des groupes fluorescents aux molécules d'agent réticulant qui sont activées par cette énergie. L'invention concerne également un procédé d'exploration de la structure tridimensionnelle de macromolécule (M) qui se caractérise en ce que: on introduit un ligand susceptible de fluorescence (F), dont la fréquence de fluorescence est comprise dans la plage 1-2, dans la macromolécule (M), ou bien en ce que l'on définit la position spatiale de ce ligand par rapport à la macromolécule (M) selon un procédé connu en soi; on provoque la liaison d'un ou de plusieurs agents réticulants (C) bifonctionnels, photoactivables, avec une fréquence d'excitation correspondante comprise dans la plage 1-2, de façon covalente, entre l'extrémité non photoactivable (S) de l'agent réticulant (C, C', C'') et des groupes fonctionnels (m) adéquats de la macromolécule (M), sans lumière; la macromolécule (M) est exposée à un rayonnement d'une fréquence Q dépassant la limite supérieure de la plage de fréquence 1-2, ce qui crée, par transfert sans rayonnement (transfert de Förster) aux agents réticulants voisins (C et/ou C''), l'activation de l'extrémité photoactivable (A et/ou A') desdits agents réticulants (C et/ou C'') produisant la réaction avec la surface de la macromolécule (M), ladite extrémité réagissant avec la surface de la macromolécule (M) en fonction de l'écart du ligand susceptible de fluorescence (F); et les groupes assemblés par paires sont identifiés selon un procédé bioanalytique, en particulier la digestion spécifique de la macromolécule (M), les fragments résultants de la digestion sont séparés, en particulier d'après la masse, et des voisinages partiaux sont déterminés par calcul .
EP99911675A 1998-02-14 1999-02-10 Activation chimique induite par transfert d'energie de fluorescence (fetma) pour l'exploration de la structure tridimensionnelle de biomacromolecules Withdrawn EP1057020A2 (fr)

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DE19806169 1998-02-14
DE19806169 1998-02-14
PCT/EP1999/001008 WO1999041607A2 (fr) 1998-02-14 1999-02-10 Activation chimique induite par transfert d'energie de fluorescence (fetma) pour l'exploration de la structure tridimensionnelle de biomacromolecules

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US (1) US6713256B1 (fr)
EP (1) EP1057020A2 (fr)
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US7368290B2 (en) * 2003-05-12 2008-05-06 Sandia National Laboratories Structural determination of intact proteins using mass spectrometry

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JP2002503810A (ja) 2002-02-05
US6713256B1 (en) 2004-03-30
WO1999041607A3 (fr) 1999-12-09

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