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Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions

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Abstract

We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9 M urea or 6 M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9 M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9 M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9 M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6 M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9 M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.

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Acknowledgements

This research was supported by a Grant from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2014R1A2A2A01002931) and the Next-Generation BioGreen 21 Program, Rural Development Administration, Republic of Korea (PJ01121601). This was also funded by the industrial research project from LG Chem.

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  1. Do Soo Jang

    Present address: Huons Co., Ltd., Seongnam, Korea

Authors and Affiliations

  1. Pohang Accelerator Laboratory, Pohang University of Science and Technology (POSTECH), Pohang, Korea

    Hyung Jin Cha & Kyeong Sik Jin

  2. Department of Life Sciences, POSTECH, Pohang, Korea

    Do Soo Jang & Kwan Yong Choi

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Handling Editor: S. Dai.

H. J. Cha and D. S. Jang contributed equally to this work.

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Cha, H.J., Jang, D.S., Jin, K.S. et al. Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions. Amino Acids 49, 715–723 (2017). https://doi.org/10.1007/s00726-016-2372-3

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