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An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates

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Peptide Self-Assembly

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1777))

Abstract

The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quantitatively interpret, without additional data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence determinations with an analytical HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.

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Acknowledgment

We acknowledge financial support from N.I.H. grant R01 GM099718.

Author information

Author notes

  1. Saketh Chemuru

    Present address: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA

  2. Pinaki Misra

    Present address: Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN, USA

  3. Ravi Kodali

    Present address: Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA

  4. Smita Mukherjee

    Present address: The Wharton School, University of Pennsylvania, Philadelphia, PA, USA

  5. Karunakar Kar

    Present address: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

Authors and Affiliations

  1. Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Ronald Wetzel, Saketh Chemuru, Pinaki Misra, Ravi Kodali, Smita Mukherjee & Karunakar Kar

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  1. Ronald Wetzel
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  2. Saketh Chemuru
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  3. Pinaki Misra
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  4. Ravi Kodali
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  6. Karunakar Kar
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Corresponding author

Correspondence to Ronald Wetzel .

Editor information

Editors and Affiliations

  1. Department of Chemistry, University of Rochester, Rochester, New York, USA

    Bradley L. Nilsson

  2. Department of Medicinal Chemistry, Institute for Translational Neuroscience, University of Minnesota, Minneapolis, Minnesota, USA

    Todd M. Doran

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Wetzel, R., Chemuru, S., Misra, P., Kodali, R., Mukherjee, S., Kar, K. (2018). An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates. In: Nilsson, B., Doran, T. (eds) Peptide Self-Assembly. Methods in Molecular Biology, vol 1777. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7811-3_6

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  • DOI: https://doi.org/10.1007/978-1-4939-7811-3_6

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7809-0

  • Online ISBN: 978-1-4939-7811-3

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