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    • UV vis spectra of hsGC

    2022-08-09

    UV–vis spectra of hsGC proteins were recorded with an HP8453 UV–vis spectrophotometer at 20°C. The corresponding ferrous, CO-bound and NO-bound species were prepared with the similar published procedure [7], [15]. The heme transfer reactions were performed by a UV–vis spectrometer with kinetic mode using the apo-myoglobin assay according to the previously published method [15].
    Results and discussion There are three cysteines (cys78, cys122 and cys174) in hsGCβ195, which are conserved in almost all eukaryotes based on the multiple sequence alignment, shown in Fig. S1 (see supporting information). Based on the homology model of hsGCβ195 published in our previous report [7], the positions of cys78, cys122 and cys174 were indicated in Fig. S2 (see supporting information). The mutants (C78A, C122A and C174S) were over-expressed in Escherichia coli and purified successfully. All the proteins are in more than 90% purity verified by 15% SDS-PAGE (Fig. 1A). According to the results of Superdex™ 200 gel filtration, shown in Fig. 1B, the wild-type hsGCβ195 and the C78A and C122A mutants mainly exist as a homodimer indicated by the column volume (∼80mL, corresponding to a molecular mass of ∼50kDa), although minor oligomers are found. However, for mutant C174S, three obvious peaks, representing oligomer, dimer and monomer, respectively, are presented. Therefore, the Darifenacin HBr of cys78 and cys122 hardly altered the aggregation states, while that of cys174 leads to less homodimerization. Herein, we demonstrated that C174 indeed contributed to the homodimerization of hsGCβ195 in a certain degree for the first time. As depicted in Fig. S2 (see supporting information), cys78 and cys122 located in α-helix, β-sheet, respectively, while cys174 was in a flexible loop. So we assumed that the mutation of cys174 would perhaps lead to the obvious conformation change through the flexible loop and accordingly alter the interaction between subunits. Admittedly, cys174 is just one influencing factor involved in the dimerization, which should be regulated by several factors cooperatively, including heme binding, hydrophobic interaction, electrostatic interaction and so on. The heme microenvironment is also closely related to the overall protein conformation. So we further study the influence of the cysteine mutations on the heme binding with UV–vis spectra and heme transfer experiments. The UV–vis spectra of the three mutants of hsGCβ195 in different forms are presented in Fig. 2, and the absorption peaks and extinction coefficients are summarized in Table S1 (see supporting information). All the forms of C122A protein exhibited similar spectral features with those of the WT hsGCβ195. As for the ferric form of C78A and C174S mutants, the Soret peak position showed about a 15nm blue-shift compared to that of WT hsGCβ195. The Soret peak of the CO-bound C174S protein also shifted to 412nm from 420nm in that of WT hsGCβ195. UV–vis observations indicated that the mutation of Cys78 and Cys174 indeed disturbed the heme microenvironment. Combined with the conclusions drew from the gel filtration results, the mutation of cys174 not only changed the aggregation state but also disturbed the heme binding microenvironment, while that of cys78 would alter the heme binding environment but not affect the aggregation. As shown in Fig. S2 (see supporting information), cys78 locates toward the vinyl side chains of heme, so it is possible that cys78 interacts with heme directly or indirectly through some other neighboring residues but is not referred to the dimerization. In the case of cys174, although far away the heme center, it is likely to affect the heme binding through conformation allosteric effect. The heme binding ability was further studied by heme transfer experiment (Fig. S3 and Table S2, see supporting information), indicating that all the three cysteine mutations showed little effect on the heme loss process.