Analytical Ultracentrifugation

The technique of analytical ultracentrifugation (AUC) was developed by Svedberg and Lysholm in 1927. Molecular weight, shape and stoichiometry of macromolecules and macromolecular complexes can be determined by AUC. AUC is an absolute method that does not require any reference or standard. 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.

Three ways to use the AUC:

  1. Sedimentation velocity centrifugation 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 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.
  2. Sedimentation equilibrium centrifugation 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; no calibration with compounds of known molecular weights is needed.
  3. Density gradient centrifugation in the analytical ultracentrifuge was once used by Meselson and Stahl to support their hypothesis about the semiconservative replication of DNA. Self-forming gradient materials, such as cesium chloride or cesium sulfate are used to establish a density gradient during centrifugation, while at the same time initially equally distributed sample molecules will form a band at the radial position where their density matches the solution density.


  • Bradshaw NJ, Yerabham ASK, Marreiros R, Zhang T, Nagel-Steger L, Korth C 
    An unpredicted aggregation-critical region of the actin-polymerizing protein TRIOBP-1/Tara, determined by elucidation of its domain structure 
    J Biol Chem 292, 9583-9598 (2017)  
  • Kroeger T, Frieg B, Zhang T, Hansen FK, Marmann A, Proksch P, Nagel-Steger L, Groth G, Smits SHJ, Gohlke H 
    EDTA aggregates induce SYPRO orange-based fluorescence in thermal shift assay 
    PLoS One 12, e0177024 (2017)  
  • Wolff M, Zhang-Haagen B, Decker C, Barz B, Schneider M, Biehl R, Radulescu A, Strodel B, Willbold D, Nagel-Steger L 
    Aβ42 pentamers/hexamers are the smallest detectable oligomers in solution 
    Sci Rep 7, 2493 (2017)  
  • Yerabham ASK, Mas PJ, Decker C, Soares DC, Weiergräber OH, Nagel-Steger L, Willbold D, Hart DJ, Bradshaw NJ, Korth C. 
    A structural organization for Disrupted in Schizophrenia 1 protein, identified by high-throughput screening, reveals distinctly folded regions which are bisected by mental illness related mutations.  
    J Biol Chem 292, 6468-6477 (2017)  
  • Luczak SE, Smits SH, Decker C, Nagel-Steger L, Schmitt L, Hegemann JH 
    The Chlamydia pneumoniae Adhesin Pmp21 Forms Oligomers with Adhesive Properties. 
    J Biol Chem 291(43), 22806-22818 (2016) 
  • Streich C, Akkari L, Decker C, Bormann J, Rehbock C, Müller-Schiffmann A, Niemeyer FC, Nagel-Steger L, Willbold D, Sacca B, Korth C, Schrader T, Barcikowski S.  
    Characterizing the Effect of Multivalent Conjugates Composed of Aβ-Specific Ligands and Metal Nanoparticles on Neurotoxic Fibrillar Aggregation. 
    ACS Nano 10, 7582-7597 (2016) 
  • Michel M, Schwarten M, Decker C, Nagel-Steger L, Willbold D, Weiergräber OH 
    The mammalian autophagy initiator complex contains 2 HORMA domain proteins 
    Autophagy 11, 2300-2308 (2015)  
  • Nouri K, Moll JM, Milroy LG, Hain A, Dvorsky R, Amin E, Lenders M, Nagel-Steger L, Howe S, Smits SH, Hengel H, Schmitt L, Münk C, Brunsveld L, Ahmadian MR 
    Biophysical Characterization of Nucleophosmin Interactions with Human Immunodeficiency Virus Rev and Herpes Simplex Virus US11. 
    PLoS One 10(12), e0143634 (2015)  
  • Wolff M, Unuchek D, Zhang B, Gordeliy V, Willbold D, Nagel-Steger L 
    Amyloid β oligomeric species present in the lag phase of amyloid formation. 
    PLoS ONE 10, e0127865 (2015)  
  • Zhao H, Ghirlando R, Alfonso C, Arisaka F, Attali I, Bain DL, Bakhtina MM, Becker DF, Bedwell GJ, Bekdemir A, Besong TM, Birck C, Brautigam CA, Brennerman W, Byron O, Bzowska A, Chaires JB, Chaton CT, Cölfen H, Connaghan KD, Crowley KA, Curth U, Daviter T, Dean WL, Díez AI, Ebel C, Eckert DM, Eisele LE, Eisenstein E, England P, Escalante C, Fagan JA, Fairman R, Finn RM, Fischle W, de la Torre JG, Gor J, Gustafsson H, Hall D, Harding SE, Cifre JG, Herr AB, Howell EE, Isaac RS, Jao SC, Jose D, Kim SJ, Kokona B, Kornblatt JA, Kosek D, Krayukhina E, Krzizike D, Kusznir EA, Kwon H, Larson A, Laue TM, Le Roy A, Leech AP, Lilie H, Luger K, Luque-Ortega JR, Ma J, May CA, Maynard EL, Modrak-Wojcik A, Mok YF, Mücke N, Nagel-Steger L, Narlikar GJ, Noda M, Nourse A, Obsil T, Park CK, Park JK, Pawelek PD, Perdue EE, Perkins SJ, Perugini MA, Peterson CL, Peverelli MG, Piszczek G, Prag G, Prevelige PE, Raynal BD, Rezabkova L, Richter K, Ringel AE, Rosenberg R, Rowe AJ, Rufer AC, Scott DJ, Seravalli JG, Solovyova AS, Song R, Staunton D, Stoddard C, Stott K, Strauss HM, Streicher WW, Sumida JP, Swygert SG, Szczepanowski RH, Tessmer I, Toth RT 4th, Tripathy A, Uchiyama S, Uebel SF, Unzai S, Gruber AV, von Hippel PH, Wandrey C, Wang SH, Weitzel SE, Wielgus-Kutrowska B, Wolberger C, Wolff M, Wright E, Wu YS, Wubben JM, Schuck P 
    A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation. 
    PLoS One 10(5), e0126420 (2015)  
  • Amin E, Dubey BN, Zhang SC, Gremer L, Dvorsky R, Moll JM, Taha MS, Nagel-Steger L, Piekorz RP, Somlyo AV, Ahmadian MR 
    Rho-kinase: regulation, (dys)function, and inhibition 
    Biol Chem. 394, 1399-1410 (2013) 
    full text  
  • Hickman DT, López-Deber MP, Ndao DM, Silva AB, Nand D, Pihlgren M, Giriens V, Madani R, St-Pierre A, Karastan H, Nagel-Steger L, Willbold D, Riesner D, Nicolau C, Baldus M, Pfeifer A, Muhs A 
    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 
    ACS Chem. Neurosci. 1, 639-648 (2010)  
    Highlighted in: Chemical & Engineering News, 88(33), August 16, 2010 
  • 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)  
  • 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)  
  • 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)  
  • 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)  
  • 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)  
  • 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)  
  • 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)  
    [This article was evaluated by the faculty of 1000 Biology as a "must read"]  
  • 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)  
  • Elfrink K, Nagel-Steger L, Riesner D 
    Interaction of the cellular prion protein with raft-like lipid membranes. 
    Biol. Chem. 388, 79-90 (2007), 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)  
  • 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)  
  • 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)  
  • 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)  
  • 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)  

Physikalische Biologie
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Prof. Dr. D. Willbold
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