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Abstract <p><b>Objective:</b> Neuroglobin and cytochrome <i>c</i> are hemoproteins whose interaction is suggested to play an important role in preventing apoptotic cell death of neurons. Therefore, studying the molecular mechanism of neuroglobin-cytochrome <i>c</i> complex formation is of significant interest. Given their small hydrodynamic size and high water solubility, these hemoproteins are well-suited for NMR spectroscopy studies, provided they are isotopically labeled with <sup>13</sup>C and <sup>15</sup>N. The aim of this work was to develop a highly efficient system for the production of <sup>15</sup>N/<sup>13</sup>C-labeled human neuroglobin and cytochrome <i>c</i>. <b>Methods:</b> The corresponding producer strains were constructed, and optimal cultivation conditions were selected, including incubation temperature and duration, medium composition, and the concentration of the expression inducer. The purified <sup>15</sup>N-labeled hemoproteins were analyzed using UV-Vis, circular dichroism (CD), and <sup>1</sup>H-<sup>15</sup>N HSQC NMR spectroscopy. <b>Results and Discussion:</b> Far- and near-UV CD spectroscopy analysis results indicated that the secondary structure composition of <sup>15</sup>N-neuroglobin is consistent with the theoretical prediction, and the heme orientation within the molecules is predominantly canonical. According to the 2D <sup>1</sup>H-<sup>15</sup>N HSQC NMR spectra of human neuroglobin and cytochrome <i>c</i>, the proteins are folded into their native conformation, characterized by a predominantly α-helical structure. <b>Conclusions:</b> An effective system for producing isotopically labeled human neuroglobin and cytochrome <i>c</i> has been developed. This system enables the preparation of high-purity <sup>15</sup>N/<sup>13</sup>C-labeled proteins suitable for structure and dynamics studies using modern high-resolution NMR spectroscopy.</p>

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Development of a Bacterial Expression System for Producing 15N/13C-Labeled Neuroglobin and Cytochrome C

  • 期刊论文

  • 发表日期：

    2025年9月28日

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  • M. A. Semenova,
  • O. M. Smirnova,
  • V. V. Britikov,
  • E. V. Britikova,
  • A. P. Khodnenko,
  • Y. V. Bershatskii,
  • A. A. Ignatova,
  • E. V. Bocharov,
  • M. P. Kirpichnikov,
  • D. A. Dolgikh,
  • R. V. Chertkova

摘要

Abstract

Objective: Neuroglobin and cytochrome c are hemoproteins whose interaction is suggested to play an important role in preventing apoptotic cell death of neurons. Therefore, studying the molecular mechanism of neuroglobin-cytochrome c complex formation is of significant interest. Given their small hydrodynamic size and high water solubility, these hemoproteins are well-suited for NMR spectroscopy studies, provided they are isotopically labeled with 13C and 15N. The aim of this work was to develop a highly efficient system for the production of 15N/13C-labeled human neuroglobin and cytochrome c. Methods: The corresponding producer strains were constructed, and optimal cultivation conditions were selected, including incubation temperature and duration, medium composition, and the concentration of the expression inducer. The purified 15N-labeled hemoproteins were analyzed using UV-Vis, circular dichroism (CD), and 1H-15N HSQC NMR spectroscopy. Results and Discussion: Far- and near-UV CD spectroscopy analysis results indicated that the secondary structure composition of 15N-neuroglobin is consistent with the theoretical prediction, and the heme orientation within the molecules is predominantly canonical. According to the 2D 1H-15N HSQC NMR spectra of human neuroglobin and cytochrome c, the proteins are folded into their native conformation, characterized by a predominantly α-helical structure. Conclusions: An effective system for producing isotopically labeled human neuroglobin and cytochrome c has been developed. This system enables the preparation of high-purity 15N/13C-labeled proteins suitable for structure and dynamics studies using modern high-resolution NMR spectroscopy.