The pneumococcal histidine triad protein D (PhtD) is believed to play a central role in pneumococcal metal ion homeostasis and has been proposed as a promising vaccine candidate against pneumococcal disease. To investigate for potential stabilizers, a panel of physiologically relevant metals was screened using the thermal shift assay and it was found that only Zn^{2+} and Mn^{2+} were able to increase PhtD melting temperature. Differential scanning calorimetry analysis revealed a sequential unfolding of PhtD and the presence of at least 3 independent folding domains that can be stabilized by Zn^{2+} and Mn^{2+}. UV spectroscopy and fluorescence quenching studies showed significant Zn^{2+}-induced tertiary structure changes in PhtD characterized by decreased accessibility of inner tryptophan residues to the aqueous solvent. Isothermal titration calorimetry data show no apparent binding to Mn^{2+} but revealed a Zn^{2+}:PhtD exothermic interaction stoichiometry of 3:1 with strong enthalpic contribution, suggesting that 3 of the 5 histidine triads are accessible binding sites for Zn^{2+}. Only Zn^{+2}, but not Mn^{+2}, was able to increase the thermal stability of PhtD in the presence of aluminum hydroxide adjuvant, making it a promising stabilizer excipient candidate in vaccine products containing PhtD.
【저자키워드】 Vaccines, stability, Spectroscopy, stabilization, biopharmaceuticals characterization, thermal analysis,