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WO2002008965A2 - Procede de determination d'ecarts spatiaux dans des polymeres ou complexes polymeres au moyen de melanges de molecules de reticulation - Google Patents

Procede de determination d'ecarts spatiaux dans des polymeres ou complexes polymeres au moyen de melanges de molecules de reticulation Download PDF

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Publication number
WO2002008965A2
WO2002008965A2 PCT/EP2001/008628 EP0108628W WO0208965A2 WO 2002008965 A2 WO2002008965 A2 WO 2002008965A2 EP 0108628 W EP0108628 W EP 0108628W WO 0208965 A2 WO0208965 A2 WO 0208965A2
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WO
WIPO (PCT)
Prior art keywords
cross
fragments
polymer
linkers
linker
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Ceased
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PCT/EP2001/008628
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German (de)
English (en)
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WO2002008965A3 (fr
Inventor
Daniel Hoffmann
Volker Schnaible
Stephan Wefing
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.)
Stiftung Caesar Center of Advanced European Studies and Research
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Stiftung Caesar Center of Advanced European Studies and Research
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Publication of WO2002008965A2 publication Critical patent/WO2002008965A2/fr
Anticipated expiration legal-status Critical
Publication of WO2002008965A3 publication Critical patent/WO2002008965A3/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the invention relates to a method for determining spatial distances in polymers or complexes of polymers, in particular biopolymers of known monomer sequences, using informatic and physicochemical methods, in particular using mixtures of cross-linker molecules.
  • the spatial structure of polymers i.e. the relative spatial arrangement of the residues, is of great importance in a large number of applications.
  • a well-known example is the rational design of new active pharmaceutical ingredients. You are looking for a small molecule that fits in the binding pocket of the target protein (such as a virus protein) like a key in a lock. The binding of such an active substance molecule to the target protein then ideally leads to the medically desired blocking of the latter. Hence, a requirement of the rational
  • the polymers have to be brought into a certain form, for example in the form of single crystals. This step is also difficult in many cases and requires lengthy series of tests.
  • cross-linkers with different lengths and end groups were used, always one cross-linker per experiment.
  • a major advantage of cross-linking methods is that comparatively small amounts of polymer are required and that no complex preparation is necessary.
  • Cross-linkers are usually somewhat flexible, so they can take an interval of lengths. The lengths determined by cross-linking are therefore uncertain. To reduce this uncertainty, several experiments can be done with overlapping
  • the object of the present invention is to provide a method for determining spatial distances in polymers which no longer has the disadvantages of the prior art mentioned.
  • the experimental effort of the method should be less than with the previously known methods, better resolution should be achieved and the method should be faster than the previous methods.
  • This task is accomplished by a spatial determination method Distances in polymers or polymer complexes of known monomer sequence solved, in which
  • the polymer molecules or complexes are reacted with mixtures of homo- or hetero-bifunctional cross-linker molecules of different lengths, masses and optionally with different chromophores with the same binding specificities of the reactive end groups,
  • the resulting fragments are identified - in particular those fragments in which cross-linkers link two residues of the polymer or complex - with the aid of a comparison of the measurement results from step c) with expected values of theoretically possible fragments based on the known ones Data on monomer sequence, cleavage sites, cross-linker, etc. are determined,
  • Proteins for example disulfide bridges
  • the length of the cross-linker from the mixture is a measure of the distance between two residues that has the shortest length among those cross-linkers of the mixture that link both residuals.
  • step a The progress of this method compared to the prior art is mainly due to two new parts of the method: firstly in the use of a mixture of cross-linkers (step a)) and secondly in the computational evaluation (step d)) of the experimental results ,
  • the method according to the invention is carried out in the following steps:
  • cross-linker molecules of different lengths.
  • the cross-linkers are chosen so that they all react approximately equally strongly with the polymer under the given conditions (pH, temperature, etc.).
  • other linkages can be used, for example naturally occurring cystines in proteins.
  • both a deuterated and a protonated version can be used in a mixture for each type of cross-linker; instead of deuteration, other isotope labels are also possible. This marking can be used to split up isobaric signals in the mass spectrum measured later, and thus facilitate the assignment of masses to molecular fragments.
  • each of the cross-linkers can be mixed with a different chromophore, so that the cross-linkers can be distinguished spectroscopically.
  • the polymer is split up specifically, that is, as precisely as possible positions given by the monomer sequence.
  • a typical example is the tryptic cleavage of polypeptides behind arginines and lysines.
  • step 3 The fragments from step 2. are separated.
  • Various methods such as high-performance liquid chromatography, liquid chromatography coupled to mass spectrometry, affinity chromatography or mass spectrometry can be used.
  • step 3 The fragments separated in step 3 are identified with parts of the polymer. A new computational procedure is used for this. Serve as input
  • g other parameters such as the maximum Number of unrecognized cleavage points or the maximum unsharpness that is accepted when comparing calculated and measured peak positions.
  • A contains all the assignments found, including all combination fragments that are compatible with the measured spectrum under the given boundary conditions.
  • B For the calculation of the base fragments, all conceivable polymer pieces between the potential cleavage sites are first generated. Parts of these polymer pieces may still be connected to one another by known connections. Each connected component from the number of conceivable polymer pieces forms its own basic fragment.
  • a stack is used to generate the non-empty subsets b of B.
  • not all possible non-empty subsets are created. For example, subsets whose mass is sufficiently far above the peak with the largest mass found in the experiment are not generated.
  • base fragments that cannot bind a cross-linker are only used for expanding the current subset if they are adjacent to at least one of the base fragments in b (and can therefore be "linked" via an unrecognized cleavage site).
  • a subset I of is "consistent” if and only if each residue from b is bound to at most one cross-linker. Exceptions are the terminal residues of the polymer, which may have more than one binding site. 5) A subset u of U is “allowed” if and only if its thickness does not exceed the predetermined maximum number of unrecognized cleavage points. 3-9) If there is only one bifunctional cross-linker type, the effort associated with steps (4), (6) and (7) can be significantly reduced. In this case it is possible to draw conclusions about the possible connection of the associated combination fragment from the thickness of the quantities and the total number of those residues in b that can bind a cross-linker.
  • the loops in steps (6) and (7) then only run over those combinations of cross-linker or cleavage numbers for which a coherent combination fragment c can be formed from b.
  • the query in step (9) is then omitted. Because the position of the cross-linker and unrecognized cleavage points in this special case at the moment of As a rule, assignment is not clearly defined, all possible related configurations are subsequently enumerated for the combination fragments from the assignments found (and only for these) at the end of the algorithm.
  • the advantages of the method according to the invention lie in the efficient use of experimental resources: the experimental effort (time, amount of polymer used) is approximately inversely proportional to the number of components of the cross-linker mixture; For example, the use of a mixture of 10 different cross-linkers reduces the experimental effort to 1/10 compared to the prior art. Effort tends to be shifted from the more expensive experimental part to the cheaper computational part.
  • ribonuclease A (RNase) from the bovine pancreas
  • RNase ribonuclease A
  • the 3D structure of this protein is already known (Howlin et al. 1989 Acta Cryst A 45: 851), so that the results of the process can be validated using an independent method.
  • the following groups can be linked by bifunctional cross-linkers to obtain distance information:
  • n 10 lysines and at the N-terminus the carboxylates on glutamates, aspartates and the C-terminus.
  • the information from the 4 disulfide bridges can be used and partially non-specifically binding photo-activatable cross-linkers can be used.
  • DMA linker length 0.86 nm
  • DMS linker length 1.10 nm
  • other cross-linkers with different linker lengths and the same reactive groups can be in the mixture.
  • the reaction product is digested with trypsin or chymotrypsin (4 hours, pH 8, 37 or 25 ° C, molar ratio enzyme / substrate 1/50).
  • the digestion is separated by reverse phase high-pressure liquid chromatography using a C18 column, 0.1% TFA in a water / acetonitrile gradient of 5-65% acetonitrile.
  • the factions are collected, dissolved in 33% acetonitrile, mixed with matrix solution (for example 10mg / ml 2,5-dihydroxybenzoic acid in 33% acetonitrile) and the peptides analyzed with matrix-assisted laser desorption / ionization mass spectrometry.
  • the mass spectra are analyzed using the algorithm described above and the linked or unlinked peptides are thereby identified. Tryptic peptides 11-33 and 86-91 (or 86-98 if modifications prevent proteolytic cleavage) appear, as well as chymotryptic peptides 31-35 and 80-97. This results in two peptides, each containing only one lysine, namely lysine 31 and 91, respectively. Via the mass, and if necessary using a post source decay mass spectrum, the two peptides or the molecule can be composed of both peptides and the linking cross Linkers can be identified without a doubt.
  • Lysines 31 and 91 are not linked by DMA or shorter cross-linkers, but they are linked by DMS and longer cross-linkers. This results in a distance between the amino groups of the two lysines of more than 1.16 nm and less than 1.40 nm (each linker length plus two bond lengths of 0.15 nm between amino-N and linker-C). So the distance is about 1.28 nm +/- 0.12 nm. In fact, there is a distance of 1.26 nm in the structure determined by X-ray crystallography, that is to say an excellent match.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de détermination d'écarts spatiaux dans des molécules polymères de séquence monomérique connue, ou dans des complexes de telles molécules polymères. Ledit procédé consiste (a) à transformer les molécules polymères ou complexes avec des mélanges de molécules de réticulation homo- ou hétéro-bifonctionnelles de longueurs et de masses différentes, et éventuellement des chromophores différents, les groupes terminaux réactifs présentant cependant les mêmes spécificités de liaison ; (b) à diviser spécifiquement les molécules polymères ou complexes après réaction avec le mélange d'agents de réticulation, au niveau de zones de division connues des séquences polymères ; (c) à mesurer les fractions ainsi obtenues, et à déterminer de préférence leur masse ; (d) à identifier les fractions obtenues, en particulier celles dans lesquelles les agents de réticulation lient deux résidus du polymère ou du complexe, au moyen d'une comparaison du résultat de mesure de l'étape (c) avec des valeurs prévisibles de fractions théoriques, lesdites valeurs étant déterminées sur la base des données connues de la séquence monomérique, des zones de rupture, des agents de réticulation, etc. ; (e) à tenir compte de liaisons présentes à l'état naturel (par exemple des ponts disulfure dans des protéines) ; et, (f) selon ledit procédé, la longueur de l'agent réticulant du mélange, présentant la plus petite longueur parmi les agents réticulants du mélange liant les deux résidus, étant une grandeur pour l'écarts de deux résidus.
PCT/EP2001/008628 2000-07-26 2001-07-26 Procede de determination d'ecarts spatiaux dans des polymeres ou complexes polymeres au moyen de melanges de molecules de reticulation Ceased WO2002008965A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2000136342 DE10036342C2 (de) 2000-07-26 2000-07-26 Verfahren zur Bestimmung räumlicher Abstände in Polymeren oder Komplexen von Polymeren mit Hilfe von Gemischen von Cross-Linker Molekülen
DE10036342.3 2000-07-26

Publications (2)

Publication Number Publication Date
WO2002008965A2 true WO2002008965A2 (fr) 2002-01-31
WO2002008965A3 WO2002008965A3 (fr) 2003-04-10

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PCT/EP2001/008628 Ceased WO2002008965A2 (fr) 2000-07-26 2001-07-26 Procede de determination d'ecarts spatiaux dans des polymeres ou complexes polymeres au moyen de melanges de molecules de reticulation

Country Status (2)

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DE (1) DE10036342C2 (fr)
WO (1) WO2002008965A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0664884A4 (fr) * 1992-05-29 1997-05-02 Univ Rockefeller Procede et produit pour la determination de sequences peptidiques a l'aide d'un spectrometre de masse.
US5453937A (en) * 1993-04-28 1995-09-26 Immunex Corporation Method and system for protein modeling
AU7146698A (en) * 1997-04-22 1998-11-13 California Institute Of Technology Method of determining three-dimensional protein structure from primary protein sequence
US6713256B1 (en) * 1998-02-14 2004-03-30 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Fluorescent energy transfer mediated chemical activation (fetma) for the elucidation of the three-dimensional structure of biomacromolecules

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Publication number Publication date
DE10036342C2 (de) 2002-09-19
DE10036342A1 (de) 2002-03-07
WO2002008965A3 (fr) 2003-04-10

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