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Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy

Nature Protocols volume 10, pages 382–396 (2015)Cite this article

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Abstract

Fourier transform IR (FTIR) spectroscopy is a nondestructive technique for structural characterization of proteins and polypeptides. The IR spectral data of polymers are usually interpreted in terms of the vibrations of a structural repeat. The repeat units in proteins give rise to nine characteristic IR absorption bands (amides A, B and I–VII). Amide I bands (1,700–1,600 cm−1) are the most prominent and sensitive vibrational bands of the protein backbone, and they relate to protein secondary structural components. In this protocol, we have detailed the principles that underlie the determination of protein secondary structure by FTIR spectroscopy, as well as the basic steps involved in protein sample preparation, instrument operation, FTIR spectra collection and spectra analysis in order to estimate protein secondary-structural components in aqueous (both H2O and deuterium oxide (D2O)) solution using algorithms, such as second-derivative, deconvolution and curve fitting. Small amounts of high-purity (>95%) proteins at high concentrations (>3 mg ml−1) are needed in this protocol; typically, the procedure can be completed in 1–2 d.

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Figure 1: Schematic overview of the full protocol.
Figure 2: Photographs of CaF2 liquid cells.
Figure 3
Figure 4: Water vapor spectrum.
Figure 5: IR analysis of hemoglobin.
Figure 6: An example of a poorly curve-fitted spectrum.
Figure 7: Second-derivative and FSD IR spectrum for three example proteins.

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Acknowledgements

This project was supported in part by grants from the National Natural Science Foundation of China (nos. 21275032, 31470786 and 21335002).

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Authors and Affiliations

  1. Department of Chemistry, Fudan University, Shanghai, China

    Huayan Yang, Shouning Yang, Jilie Kong & Shaoning Yu

  2. Department of Chemistry and Biochemistry, University of Northern Colorado, Greeley, Colorado, USA.,

    Aichun Dong

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Contributions

H.Y. and S. Yu designed, implemented and wrote the protocol. S. Yang and J.K. implemented the protocol. A.D. contributed to data analysis.

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Correspondence to Aichun Dong or Shaoning Yu.

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Yang, H., Yang, S., Kong, J. et al. Obtaining information about protein secondary structures in aqueous solution using Fourier transform IR spectroscopy. Nat Protoc 10, 382–396 (2015). https://doi.org/10.1038/nprot.2015.024

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