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<p>Rotaviruses (RV) are a major cause of severe childhood diarrhoea, particularly in developing nations, necessitating stable vaccines. Therefore, the presented preliminary study aimed to assess the impact of altered physicochemical properties on the structural stability of recombinant rotavirus capsid protein VP6 (RV-VP6). The expression system used in this study was designed by genetically engineering the RV-VP6 into <i>E. coli</i> (NiCo21(DE3))-pET28a host-vector system and purified using liquid chromatography. The purified RV-VP6 homology detection and structure prediction were conducted using LC–MS and HHpred computational analysis, which indicated a 100% probability of 1QHD_A Viral Capsid VP6 (1.95&#xa0;Å), representing the crystal structure of VP6. The secondary and tertiary structural stability of RV-VP6 was evaluated in altered pH and Ca<sup>2+</sup> concentrations using far UV-CD and intrinsic tryptophan fluorescence spectroscopy, respectively. The computational analysis of the far-UV CD spectra revealed a significant increase in the composition of α-helices and β-sheets in altered pH and Ca<sup>2+</sup> environments compared to the denatured protein (<i>p</i> &lt; 0.0001). Intrinsic fluorescence analysis of RV-VP6 at pH 7 yielded an emission λmax of 339&#xa0;nm, which shifted to 342&#xa0;nm at pH 5. In 1&#xa0;mM Ca<sup>2+</sup>, a λmax of 340&#xa0;nm was observed, with an increase in intensity in 10&#xa0;mM Ca<sup>2+</sup>, accompanied by a slight blue shift to 338&#xa0;nm. Investigation of RV-VP6 under thermal stress yielded unfolding concomitant with aggregation, rendering the process irreversible and nullifying analysis using equilibrium thermodynamics. These findings form the preliminary basis for our future evaluation of manufacturing stable and enhanced RV-VP6 vaccines through the downstream process control of (1) pH, which alters the charge distribution on the surface of the protein, leading to conformational changes, and (2) Ca<sup>2+</sup> ions, which interact with specific amino acid residues in the protein, thereby affecting its structure and function.</p>

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Evaluating the Structural Stability of Recombinant Rotavirus Capsid Protein VP6 in Altered Physicochemical States Using Fluorescence and CD Spectroscopy

  • 期刊论文

  • 发表日期：

    2025年11月16日

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  • Modibedi Lesego Gladys,
  • Preenan Pillay

摘要

Rotaviruses (RV) are a major cause of severe childhood diarrhoea, particularly in developing nations, necessitating stable vaccines. Therefore, the presented preliminary study aimed to assess the impact of altered physicochemical properties on the structural stability of recombinant rotavirus capsid protein VP6 (RV-VP6). The expression system used in this study was designed by genetically engineering the RV-VP6 into E. coli (NiCo21(DE3))-pET28a host-vector system and purified using liquid chromatography. The purified RV-VP6 homology detection and structure prediction were conducted using LC–MS and HHpred computational analysis, which indicated a 100% probability of 1QHD_A Viral Capsid VP6 (1.95 Å), representing the crystal structure of VP6. The secondary and tertiary structural stability of RV-VP6 was evaluated in altered pH and Ca2+ concentrations using far UV-CD and intrinsic tryptophan fluorescence spectroscopy, respectively. The computational analysis of the far-UV CD spectra revealed a significant increase in the composition of α-helices and β-sheets in altered pH and Ca2+ environments compared to the denatured protein (p < 0.0001). Intrinsic fluorescence analysis of RV-VP6 at pH 7 yielded an emission λmax of 339 nm, which shifted to 342 nm at pH 5. In 1 mM Ca2+, a λmax of 340 nm was observed, with an increase in intensity in 10 mM Ca2+, accompanied by a slight blue shift to 338 nm. Investigation of RV-VP6 under thermal stress yielded unfolding concomitant with aggregation, rendering the process irreversible and nullifying analysis using equilibrium thermodynamics. These findings form the preliminary basis for our future evaluation of manufacturing stable and enhanced RV-VP6 vaccines through the downstream process control of (1) pH, which alters the charge distribution on the surface of the protein, leading to conformational changes, and (2) Ca2+ ions, which interact with specific amino acid residues in the protein, thereby affecting its structure and function.