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

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.