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Site-Directed Mutagenesis of BmK AGP-SYPU1: The Role of Two Conserved Tyr (Tyr5 and Tyr42) in Analgesic Activity

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Abstract

In this study, the role of two conversed tyrosines (Tyr5 and Tyr42) from the scorpion toxin BmK AGP-SYPU1 was investigated with an effective Escherichia coli expression system. Site-directed mutagenesis was used to individually substitute Tyr5 and Tyr42 with hydrophobic or hydrophilic amino acids, and the extent to which these scorpion toxin BmK AGP-SYPU1 tyrosines contribute to analgesic activity was evaluated. The results of the mouse-twisting test showed that Tyr5 and Tyr42 are associated with the analgesic activity of the toxin because the analgesic activities of Y5F and Y42F were significantly increased compared with the rBmK AGP-SYPU1; however, the Y5W had decreased activity. The results of molecular simulation reveal the following: (1) for analgesic activity, the core domain of the scorpion toxin BmK AGP-SYPU1 is key and (2) for pharmacological function, Tyr42 is most likely involved when the core domain conformation is altered. These studies identify a new relationship between the structure and analgesic activity of the scorpion toxin BmK AGP-SYPU1 and are significant for further research and the application of analgesic peptides.

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Abbreviations

BmK:

Buthus martensii Karsch

BmK AGP-SYPU1:

Analgesic peptide 1 from Buthus martensii Karsch by Shenyang Pharmaceutical University

rBmK AGP-SYPU1:

Recombinant BmK AGP-SYPU1

Lqq:

Leiurus quinquestriatus quinquestriatus

E. coli :

Escherichia coli

PCR:

Polymerase chain reaction

3D:

Three-dimensional

SDS:

Sodium dodecyl sulfate

PAGE:

Polyacrylamide gel electrophoresis

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Acknowledgments

This study was supported by the Grants from the National Natural Science Foundation of China (81072568), Major Drug Innovation of National Eleventh Five-Year Major Project of China (2009ZX09301-012), the Natural Science Foundation of China (81102365), and Specialized Research Fund for the Doctoral Program of Higher Education (20112134120008).

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Authors and Affiliations

  1. School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016, Liaoning Province, People’s Republic of China

    Li Deng, Hong-Xia Zhang, Rong Zhang, Xue Wen, Yong-Bo Song, Yong-Shan Zhao, Lin Ma, Chun-Fu Wu & Jing-Hai Zhang

  2. College of Life Science and Bioengineering, Shenyang University, Shenyang, 110044, Liaoning Province, People’s Republic of China

    Li Deng

  3. The 463rd Hospital of PLA, Shenyang, 110042, Liaoning Province, People’s Republic of China

    Yu Wang

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  1. Li Deng
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  2. Hong-Xia Zhang
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  3. Yu Wang
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  8. Lin Ma
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Correspondence to Jing-Hai Zhang.

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Deng, L., Zhang, HX., Wang, Y. et al. Site-Directed Mutagenesis of BmK AGP-SYPU1: The Role of Two Conserved Tyr (Tyr5 and Tyr42) in Analgesic Activity. Protein J 33, 157–164 (2014). https://doi.org/10.1007/s10930-014-9547-0

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