Abstract
This chapter provides protocols for isotope-labeling the human islet amyloid polypeptide (hIAPP or amylin) involved in type II diabetes and γD-crystallin involved in cataract formation. Because isotope labeling improves the structural resolution, these protocols are useful for experiments using Fourier transform infrared (FTIR), two-dimensional infrared (2D IR), and NMR spectroscopies. Our research group specializes in using 2D IR spectroscopy and isotope labeling. 2D IR spectroscopy provides structural information by measuring solvation from 2D diagonal lineshapes and vibrational couplings from cross peaks. Infrared spectroscopy can be used to study kinetics, membrane proteins, and aggregated proteins. Isotope labeling provides greater certainty in the spectral assignment, which enables new structural insights that are difficult to obtain with other methods. For amylin, we provide a protocol for 13C/18O labeling backbone carbonyls at one or more desired amino acids in order to obtain residue-specific structural resolution. We also provide a protocol for expressing and purifying amylin from E. coli, which enables uniform 13C or 13C/15N labeling. Uniform labeling is useful for measuring the monomer infrared spectrum in an amyloid oligomer or fiber as well as amyloid protein bound to another polypeptide or protein, such as a chaperone or an inhibitor. In addition, our expression protocol results in 2–2.5 mg of amylin peptide per 1 L cell culture, which is a high enough yield to straightforwardly obtain the 2–10 mg needed for high resolution and solid-state NMR experiments. Finally, we provide a protocol to isotope-label either of the two domains of γD-crystallin using expressed protein ligation. Domain labeling makes it possible to resolve the structures of the two halves of the protein in FTIR and 2D IR spectra. With modifications, these strategies and protocols for isotope labeling can be applied to other amyloid polypeptides and proteins.
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Acknowledgments
We thank NIH NIDDK DK79895 for supporting this work.
Statement: Martin Zanni is a cofounder of PhaseTech Spectroscopy, Inc. which manufactures 2D IR spectrometers.
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Zhang, T.O., Grechko, M., Moran, S.D., Zanni, M.T. (2016). Isotope-Labeled Amyloids via Synthesis, Expression, and Chemical Ligation for Use in FTIR, 2D IR, and NMR Studies. 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_2
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DOI: https://doi.org/10.1007/978-1-4939-2978-8_2
Publisher Name: Humana Press, New York, NY
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