Abstract
The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Insights into the prerequisites and kinetics of the polymerization was obtained by the in vitro analysis of this process. In the past, fluorescent dyes were used to stain amyloidogenic material in histology and later on similar dyes were used in in vitro studies as well. To circumvent the flaws of extragenous dyes, namely the alteration of the polymerization kinetic or incompatibility with other chemical compounds needed for stability analysis, we applied tryptophan fluorescence to the in vitro analysis of PHF formation. Single tryptophans were introduced into the hexapeptide PHF6 within the third repeat, which was shown to be involved in ß sheet formation and scattered around the whole microtubule binding domain. Tryptophan fluorescence was then used to scan the microtubule binding domain for accessibility to quenching reagent in the soluble and the aggregated state and the fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine 310. Furthis approach enables the analysis of stability of PHFs in the presence of Guanidinium hydrochloride. The examples given here could be applied in modified ways to other amyloidogenic proteins.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Taylor, J. P., Hardy, J., and Fischbeck, K. H. (2002) Toxic proteins in neurode-generative disease. Science 296, 1991–1995.
Selkoe, D. J. (2003) Folding proteins in fatal ways. Nature 426, 900–904.
Johnson, G. V. and Bailey, C. D. (2002) Tau, where are we now? J. Alzheimers Dis. 4, 375–398.
Garcia, M. L. and Cleveland, D. W. (2001) Going new places using an old MAP: tau, microtubules and human neurodegenerative disease. Curr. Opin. Cell Biol. 13, 41–48.
Goedert, M., Wischik, C. M., Crowther, R. A., Walker, J. E., and Klug, A. (1988) Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau. Proc. Natl. Acad. Sci. USA 85, 4051–4055.
Hutton, M. (2000) Molecular genetics of chromosome 17 tauopathies. Ann. NY Acad. Sci. 920, 63–73.
Goedert, M., Ghetti, B., and Spillantini, M. G. (2000) Tau gene mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Their relevance for understanding the neurogenerative process. Ann. NY Acad. Sci. 920, 74–83.
Glenner, G. G., Eanes, E. D., and Page, D. L. (1972) The relation of the properties of Congo red-stained amyloid fibrils to the ß-conformation. J. Histochem. Cytochem. 20, 821–826.
Vallet, P. G., Guntern, R., Hof, P. R., et al. (1992) A comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in Alzheimer's disease. Acta Neuropathol. (Berl) 83, 170–178.
LeVine, H. (1993) Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 2, 404–410.
Sipe, J. D. and Cohen, A. S. (2000) Review: history of the amyloid fibril. J. Struct. Biol. 130, 88–98.
Kirschner, D. A., Teplow, D. B., and Damas, A. M. (2000) Twist and sheet: variations on the theme of amyloid. J. Struct. Biol. 130, 87.
Findeis, M. A. (2000) Approaches to discovery and characterization of inhibitors of amyloid beta-peptide polymerization. Biochim. Biophys. Acta 1502, 76–84.
Kuner, P., Bohrmann, B., Tjernberg, L. O., et al. (2000) Controlling polymerization of beta-amyloid and prion-derived peptides with synthetic small molecule ligands. J. Biol. Chem. 275, 1673–1678.
Braak, H., Braak, E., Ohm, T., and Bohl, J. (1989) Alzheimer's disease: mismatch between amyloid plaques and neuritic plaques. Neurosci. Lett. 103, 24–28.
Friedhoff, P., Schneider, A., Mandelkow, E. M., and Mandelkow, E. (1998) Rapid assembly of Alzheimer-like paired helical filaments from microtubule-associated protein tau monitored by fluorescence in solution. Biochemistry 37, 10223–10230.
von Bergen, M., Friedhoff, P., Biernat, J., Heberle, J., Mandelkow, E. M., and Mandelkow, E. (2000) Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc. Natl. Acad. Sci. USA 97, 5129–5134.
Friedhoff, P., von Bergen, M., Mandelkow, E. M., and Mandelkow, E. (1998) A nucleated assembly mechanism of Alzheimer paired helical filaments. Natl. Acad. Sci. USA 95, 15712–15717.
Biernat, J., Mandelkow, E. M., Schröter, C., et al. (1992) The switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serine-proline motifs upstream of the microtubule binding region. EMBO J. 11, 1593–1597.
Barghorn, S., Zheng-Fischhofer, Q., Ackmann, M., Biernat, J., von Bergen, M., and Mandelkow, E. (2000) Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias. Biochemistry 39, 11714–11721.
Eftink, M. R. (1994) The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys. J. 66, 482–501.
Li, L., von Bergen, M., Mandelkow, E. M., and Mandelkow, E. (2002) Structure, stability, and aggregation of paired helical filaments from tau protein and FTDP-17 mutants probed by tryptophan scanning mutagenesis. J. Biol. Chem. 277,41390–41400.
von Bergen, M., Barghorn, S., Li, L., Marx, A., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (2001) Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure. J. Biol. Chem. 276,48165–48174.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
von Bergen, M., Li, L., Mandelkow, E. (2005). Intrinsic Fluorescent Detection of Tau Conformation and Aggregation. In: Sigurdsson, E.M. (eds) Amyloid Proteins. Methods in Molecular Biology™, vol 299. Humana Press. https://doi.org/10.1385/1-59259-874-9:175
Download citation
DOI: https://doi.org/10.1385/1-59259-874-9:175
Publisher Name: Humana Press
Print ISBN: 978-1-58829-337-4
Online ISBN: 978-1-59259-874-8
eBook Packages: Springer Protocols