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Analytische Ultrazentrifugation

Die Technik der analytischen Ultrazentrifugation (AUZ) wurde durch Svedberg und Lysholm 1927 entwickelt (1). Mit AUZ läßt sich das Molekulargewicht, die Form und die Stöchiometrie von Makromolekülen und makromolekularen Komplexen bestimmen.

Die AUZ ist eine absolute Methode, welche keinen Vergleichsstandard erfordert. Obwohl neuere Methoden wie die Gelelektrophorese oder die Gelfiltration einige Anwendungsgebiete der AUZ ersetzt haben.... Though methods like gel electrophoresis or size exclusion chromatography replaced part of the applications of AUC, nowadays AUC experiences a renaissance based on the need for information about the oligomeric state or conformation of proteins under physiological conditions, which cannot be retrieved from the knowledge of the amino acid sequence of the single polypeptide chain alone. Additionally the utilisation of computer aided data acquisition and data evaluation disclosed new perspectives in the amount of information that can be retrieved from analytical ultracentrifugation experiments.

The method offers the opportunity to retrieve structural information about biological macromolecules in solution for a broad concentration range and under a wide variety of solvent conditions. The hydrodynamic behaviour of macromolecules under the influence of centrifugal forces is determined by their molecular size and molecular shape and the physical and chemical properties of the solvent.

Two different experimental protocols are used:

  1. Sedimentation equilibrium centrifugation (SEC) is utilized to determine the molecular weight of a molecule or a molecular complex. By prolonged centrifugation which leads to the establishment of an equilibrium between sedimentation and back diffusion of the particles a time-independent concentration profile is obtained from which the molecular weight can be extracted. AUC is an absolute method regarding the molecular weight determination because no calibration with compounds of known molecular weights is needed.
  2. Sedimentation velocity centrifugation (SVC) or moving boundary sedimentation is utilized to determine sedimentation coefficients or sedimentation coefficient distributions by observation of the particle behaviour during the sedimentation process. Under the influence of the applied centrifugational field in an initially uniformly filled sample cell a boundary (Fig. 1, right) is formed between an area depleted of the sedimenting molecules near the rotor axis and the remaining solution. The motion of this boundary over time is a measure for the velocity of the sedimenting molecules. From the sedimentation velocity molecular properties like size and shape of a particle can be infered.

The instrument, an Optima XL-A from Beckman-Coulter (Fig1, left), allows for sedimentational speeds from 3000 to 60,000 rpm generating up to 250,000 fold acceleration of gravity in the centre of the sample cell. The instrument is equipped with an absorption optics, allowing measurements in the spectral range between 190 and 600 nm with a sensitivity of 0.3 to 0.9 OD for SVC and 0.1 to 0.3 OD for SEC.

(1) An Ultracentrifuge of oil turbine type for the determination of molecular weights : Contribution from the laboratory of physical chemistry of the University of Upsala / The. Svedberg ; Alf Lysholm: Nova Acta Regiae Societatis scientiarum Upsaliensis ; Ser. 4, vol. extra ord. 1927, [14]

Artikel 1

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    Rho-kinase: regulation, (dys)function, and inhibition
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    Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases.
    J. Biol.Chem. 286, 13966-13976 (2011)
  • Funke SA, van Groen T, Kadish I, Bartnik D, Nagel-Steger L, Brener O, Sehl T, Batra-Safferling R, Moriscot C, Schoehn G, Horn AHC, Müller-Schiffmann A, Korth C, Sticht H, Willbold D
    Oral Treatment with the D-Enantiomeric Peptide D3 Improves Pathology and Behavior of Alzheimer?s disease Transgenic Mice
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    http://dx.doi.org/10.1021/cn100057j
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  • Merdanovic M, Mamant N, Meltzer M, Poepsel S, Auckenthaler A, Melgaard R, Hauske P, Nagel-Steger L, Clarke AR, Kaiser M, Huber R, Ehrmann M
    Determinants of structural and functional plasticity of a widely conserved protease chaperone complex.
    Nat. Struct. Mol. Biol. 17, 837-843 (2010)
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  • Müller-Schiffmann A, März-Berberich J, Andrjevna A, Rönicke R, Bartnik D, Brener O, Kutzsche J, Horn AHC, Hellmert M, Polkowska J, Gottmann K, Reymann K, Funke SA, Nagel-Steger L, Moriscot C, Schoehn G, Sticht H, Willbold D, Schrader T, Korth C
    Combining independent drug classes into superior, synergistically acting hybrid molecules.
    Angew. Chem. Int. Ed. Engl. 49, 8743-8746 (2010)
    http://dx.doi.org/10.1002/anie.201004437
  • Nagel-Steger L, Demeler B, Meyer-Zaika W, Hochdörffer K, Schrader T, Willbold D
    Modulation of aggregate size- and shape-distributions of the amyloid-beta peptide by a designed ß-sheet breaker.
    Eur. Biophys. J. 39, 415-422 (2010)
    http://dx.doi.org/10.1007/s00249-009-041
  • Demeler B, Brookes E, Nagel-Steger L
    Analysis of heterogeneity in molecular weight and shape by analytical ultracentrifugation using parallel distributed computing.
    Meth. Enzymol. 454, 87-113 (2009)
    http://dx.doi.org/10.1016/S0076-6879(08)03804-4
  • Nagel-Steger L, Demeler B, Hochdörfer K, Schrader T, Willbold D
    Modulation of aggregate size and shape distributions of amyloid-ß peptide solutions by a designed ß-sheet breaker.
    NIC Workshop 2008 (From Computational Biophysics to Systems Biology; CBSB08) Vol. 40, (2008)
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  • Stöhr J, Weinmann N, Wille H, Kaimann T, Nagel-Steger L, Birkmann E, Panza G, Prusiner SB, Eigen M, Riesner D
    Mechanisms of prion protein assembly into amyloid.
    Proc. Natl. Acad. Sci. U S A 105, 2409-2414 (2008)
    http://dx.doi.org/10.1073/pnas.0712036105
  • van Groen T, Wiesehan K, Funke SA, Kadish I, Nagel-Steger L, Willbold D
    Reduction of Alzheimer's disease amyloid plaque load in transgenic mice by D3, a D-enantiomeric peptide identified by mirror image phage display.
    ChemMedChem 3, 1848-1852 (2008)
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  • Wiesehan K, Stöhr J, Nagel-Steger K, van Groen T, Riesner D and Willbold D
    Inhibition of cytotoxicity and fibril formation by a D-amino acid peptide that specifically binds to Alzheimer's disease amyloid peptide
    Prot. Eng. Des. Sel. 21, 241-246 (2008)
    http://dx.doi.org/10.1093/protein/gzm054
  • Elfrink K, Nagel-Steger L, Riesner D
    Interaction of the cellular prion protein with raft-like lipid membranes.
    Biol. Chem. 388, 79-90 (2007)
    ttp://dx.doi.org/10.1515/BC.2007.010, 01/01/2007
  • Muhs A, Hickmann DT, Pihlgren M, Chuard N, Giriens V, Meerschman C, van der Auwera I, van Leuven F, Sugawara M, Weingertner MC, Bechinger B, Greferath R, Kolonko N, Nagel-Steger L, Riesner D, Brady RO, Pfeifer A, Nicolau C
    Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice
    Proc. Natl. Acad. Sci. 104, 9810-9815 (2007)
    http://dx.doi.org/10.1073/pnas.0703137104
  • Nagel-Steger L, Demeler B, Willbold D
    Aggregate size and shape distributions in amyloid-ß peptide solutions. Aggregate Size and Shape Distributions in Amyloid-beta Peptide Solutions.
    NIC Workshop (From Computational Biophysics to Systems Biology; CBSB07) Vol. 36, (2007)
    http://www.fz-juelich.de/nic-series/volume36/nagel-steger.pdf
  • Riesner, D, Birkmann E, Dumpitak C, Elfrink K, Kellings K, Leffers K-W, Nagel-Steger L, Stöhr J
    PrP-Fibrillen und Infektiosität
    Nova Acta Leopoldina 347, 61-77 (2006)
  • Leffers KW, Schell J, Jansen K, Lucassen R, Kaimann T, Nagel-Steger L, Tatzelt J, Riesner D
    The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites.
    J Mol Biol 344, 839-853 (2004)
    http://dx.doi.org/10.1016/j.jmb.2004.09.071
  • Rzepecki P, Nagel-Steger L, Feuerstein S, Linne U, Molt O, Zadmard R, Aschermann K, Wehner M, Schrader T, Riesner D
    Prevention of Alzheimer's disease-associated Aß aggregation by rationally designed nonpeptidic ß-sheet ligands.
    J Biol Chem 279, 47497-47505 (2004)
    http://dx.doi.org/10.1074/jbc.M405914200
  • Böttcher B, Scheide D, Hesterberg M, Nagel-Steger L, Friedrich T
    A novel, enzymatically active conformation of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I).
    J Biol Chem 277, 17970-17977 (2002)
    http://dx.doi.org/10.1074/jbc.M112357200

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