Fig 1.
Structure and sequence comparison.
(A) Enlarged view of the aromatic cluster side of SpGrx3 and (B) SpTrx. (C) Overlap structures of SpGrx3 (gold) and SpTrx (blue), highlighting the additional α-helix and β-strand in SpTrx (purple). (D) Sequence comparison of Trx and Grx3 members with different habitat temperatures. The active site motif is denoted by the cyan box. The α-helices and β-strands of Grx3 and Trx are labeled with superscripts G and T, respectively. The sequences examined include SpGrx3 (WP_010217562.1), PhGrx3 (YP_338909.1), CpGrx3 (WP_011045119.1), SpTrx (WP_010164143.1), PhTrx (WP_008109071), CpTrx (KGJ92637.1), EcGrx3 (1FOV), RnGrx3 (WP_008217797.1), AfGrx3 (WP_035195182.1), EcTrx (2TRX), RnTrx (WP_008222599.1), AfTrx (AQS23897.1), PeGrx3 (TXF13737.1), PtGrx3 (WP_191130001.1), TtGrx3 (QGU33019.1), LBCA (4BA7), TtTrx (BAW01746.1), and TeTrx (EEU61346.1).
Fig 2.
Size-exclusion chromatography analysis.
Determination of the MW of native SpGrx3 WT and mutants by size-exclusion chromatography on a Superdex 200 prep grade XK16 column. The arrows indicate that the estimated MW of SpGrx3 WT and other mutants is 11.0 kDa, while the R47F mutant forms a dimer with an MW of 18.0 kDa.
Fig 3.
Intrinsic fluorescence and acrylamide Stern–Volmer plot of SpGrx3 WT and mutants.
(A) Intrinsic fluorescence at 25°C (excitation at 275 nm). The fluorescence of the WT was normalized to 100%. (B) Acrylamide Stern–Volmer plot. F0 represents the maximum fluorescence intensity without acrylamide, while F represents the maximum fluorescence intensity at increasing concentrations (0–0.25 M) of acrylamide. Data presented are the means ± S.D. of three experiments.
Table 1.
Tm values of SpGrx3 WT and mutants.
Fig 4.
Bis-ANS fluorescence of SpGrx3 WT and mutants.
The temperature-induced unfolding of SpGrx3 and its mutants was assessed by measuring bis-ANS fluorescence after incubating the proteins at various temperatures (4–60°C) for 1 h (excitation at 385 nm). The highest fluorescence of each protein was set as 100%. Data presented are the means of three experiments.
Fig 5.
The urea-induced unfolding of SpGrx3 WT and mutants.
Fluorescence spectra were measured after incubating the proteins with different concentrations of urea (0–9 M) at 25°C for 20 min (excitation at 275 nm). Data presented are the means ± S.D. of three experiments.
Table 2.
Stability parameters for SpGrx3 WT and mutants.
Fig 6.
Far UV-CD spectra of SpGrx3 WT and mutants.
The CD spectra were measured at 25°C following a 1-h incubation of the proteins at 4°C.
Table 3.
Kinetic parameters of SpGrx3 WT and mutants.