Abstract
Amyloid fibrils are associated with a number of human diseases. These aggregatively misfolded intermolecular β-sheet assemblies constitute some of the most challenging targets in structural biology because to their complexity, size, and insolubility. Here, protocols and controls are described for experiments designed to study hydrogen-bonding in amyloid fibrils indirectly, by transferring information about amide proton occupancy in the fibrils to the dimethyl sulfoxide-denatured state. Since the denatured state is amenable to solution NMR spectroscopy, the method can provide residue-level-resolution data on hydrogen exchange for the monomers that make up the fibrils.
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Abbreviations
- DCA:
-
Dichloroacetic acid
- DCA-d2:
-
Deuterated analog of dichloroacetic acid: Cl2CDCO2D
- DCl:
-
Deuterium chloride (deuterated analog of HCl)
- DMSO:
-
Dimethyl sulfoxide
- DMSO-d6:
-
Deuterated analog of dimethyl sulfoxide (CD3)2SO
- D2O:
-
Deuterium oxide (heavy water)
- HSQC:
-
Heteronuclear single-quantum coherence
- IAPP:
-
Islet amyloid polypeptide
- NMR:
-
Nuclear magnetic resonance
- NOESY:
-
Nuclear Overhauser enhancement spectroscopy
- qHX:
-
Quenched hydrogen exchange
- SDS:
-
Sodium dodecyl sulfate
- ThT:
-
Thioflavin T
- TOCSY:
-
Total correlation spectroscopy
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Acknowledgments
This work was supported by Basic Research Award 1-10-BS-04 to A.T.A. from the American Diabetes Association. I thank Rebecca L. Newcomer and Anne R. Kaplan for useful comments on the manuscript.
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Alexandrescu, A.T. (2016). Quenched Hydrogen Exchange NMR of Amyloid Fibrils. In: Eliezer, D. (eds) Protein Amyloid Aggregation. Methods in Molecular Biology, vol 1345. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2978-8_14
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DOI: https://doi.org/10.1007/978-1-4939-2978-8_14
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