Table 1.
Indication of the FGF2 name described in the manuscript.
Fig 1.
A comparative analysis of sequence and thermal stability in human and whale FGF2.
(A) Mutation positions are indicated with yellow boxes and red text. The different sequences of humans and whales are indicated with a black star. Beta-strands are indicated by a black arrow. Protein sequence alignment was calculated using ESPript 3.0 (Robert and Gouet 2014) and MultAlin (Corpet 1988) servers. (B) The FGF2 mutants (T121S, S137P, T121S/S137P) with whale sequences are tested for thermal stability for six days at 37 °C, compared with wild-type human FGF2. Protein stability is demonstrated by SDS-PAGE gel and (C) Reverse phase HPLC. FGF2 variants (FGF2, FGF2 T121S, FGF2 S137P, FGF2 T121S/S137P) were tested for 6 days. The main protein bands of the FGF2 variants (FGF2 (▤), FGF2 T121S (▥), FGF2 S137P (■), and FGF2 T121S/S137P (▧) are calculated as percentages and shown as bar graphs.
Fig 2.
Glutathione effect of FGF2 mutants.
(A) The result of 15% SDS-PAGE after incubating pFGF2 with 5 mM glutathione at 45 °C for 6 days. The bands analyzed by GelNrich-coupled mass spectrometry are marked in red boxes and labeled as ①, ②, and ③. The pFGF2 sequence is shown in the right box, with D15 and D57 indicated in red text. The major fragments of pFGF2 analyzed by N-terminal sequencing are highlighted in gray and labeled as ①, ②, and ③. (B) The structure of FGF2 with D15, D57 (green sticks), and D28 (a magenta stick). (C) The result of 15% SDS-PAGE after incubating pFGF2, pFGF2 D15E, and pFGF2 D28E with 0 ~ 20 mM GSSG and GSH, respectively.
Table 2.
Prediction of FGF2 stability using SDM.
Table 3.
Prediction of FGF2 stability using Discovery studio 2019.
Fig 3.
Structural comparison of FGF2-M1 and the FGF2-M2/SOS complex.
Left, The superimposed Cα tracing of the FGF2 wild type (yellow; PDB code: 4FGF), the structure of the FGF2 mutant harboring C78S & C96S (orange; PDB code: 1BFG), FGF2-M1 (green), and the FGF2-M2/SOS complex (purple). The N- and C-terminus of FGF2-M1 are shown as blue and red spheres, respectively. Right, Surface representation with electrostatic potentials of FGF2-M2 with SOS (purple stick). The positively and negatively charged regions on the surface are colored in blue and red, respectively. In the box, the SOS-interacting residues of FGF2-M2 are shown as white sticks with labels. Nitrogen, oxygen, and sulfur atoms in the sticks are colored in blue, red, and yellow, respectively.
Fig 4.
The mutation sites in the crystal structures of FGF2-M1 and FGF2-M2.
(A) A side view & a top view of cartoon diagrams of FGF2 mutants labeled with each β strand in a β-barrel domain (green) and a triangular cap domain (gray). For brevity, only a cartoon diagram of the FGF2-M2/SOS complex is presented while the mutations in both structures of FGF2-M1 (pink sticks) and FGF2-M2 (purple sticks) are represented. (B-D) Close-up view of the mutation sites. The interacting residues with the mutation sites are shown as labeled sticks. The arrows indicate the β strands. Nitrogen, oxygen, and sulfur atoms in the sticks are colored blue, red, and yellow, respectively. (C) The surfaces of C78L, C78I, and interacting residues (P45 & P58 & H59 & L77) are represented as dots. (D) Interactions between C96I, T98, adjacent residues (K86 & E100), and a water molecule (a red sphere) are depicted by dashed lines with labeled distances (Å).
Fig 5.
Thermal denaturation curve measured by CD experiments.
Each graph displays the temperature-dependent spectral change at 228 nm. FGF2 and the mutants are represented by different colored lines and symbols, as shown in the right box of the figure.
Fig 6.
Comparison of protease digestion profiles of FGFs.
Six different proteases were mixed with FGF2 variants (FGF2, FGF2 D28E, FGF2 C78S/C96S, FGF2-M1, and FGF2-M2). The protease reaction was conducted at 37 °C for 3 hours. The remaining proteins identified in the SDS-PAGE gel were quantified using the ImageJ program. The quantified values were presented as a bar graph. Cleaved proteins were excluded from quantification. The horizontal axis of the graph represents the following order: unincubated FGF2, incubated FGF2, trypsin, subtilisin, protease K, actinase, elastase, and papain. The vertical axis of the graph represents the percentage of protease digestion (N = 3, average ± SD). The statistical differences at each protease were performed by one-way ANOVA with Tukey’s post hoc test. Asterisk (*) indicates P < 0.05.
Fig 7.
Cell proliferation activity of FGF2 mutants.
(A) BALB3T3 cells were treated with 0.3 ng/mL FGF2s for 40 h, and then the proliferation assay was performed using cell counting kit-8. The proliferation levels of non-treated cells (Control) were set to 1, and that of the treated cells was calculated relative to this value (n = 4, average ± SD). The statistical differences were analyzed using the one-way ANOVA, followed by Tukey’s post hoc test. *P < 0.05 vs. Control. n.s., not significant. (B, C) After incubation of FGF2s at 37 °C (B) or 45 °C (C) for the indicated duration, their residual activity was determined using the cell proliferation assay. BALB3T3 cells were treated with 0.3 ng/mL FGF2s for 40 h, followed by the cell proliferation assay. The proliferation levels of non-incubated FGF2 at 37 °C (B) or 45 °C (C) were set to 100%, and that of the treated cells was calculated relative to this value (n = 3, average ± SD). Non-linear regression was performed using GraphPad Prism 9.4, and the best-fit non-linear regression curve is depicted. The statistical differences at each time point were calculated using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05. The dotted lines indicate half-levels of proliferation and their incubation day.