Protein conformation stabilized by newly formed turns for thermal resilience

Abstract

Thermally stable or resilient proteins are usually stabilized at intermediate states during thermal stress to prevent irreversible denaturation. However, the mechanism by which their conformations are stabilized to resist high temperature remains elusive. Herein, we investigate the conformational and thermal stability of transforming growth factor-β1 (TGF-β1), a key signaling molecule in numerous biological pathways. We report that the TGF-β1 molecule is thermally resilient as it gradually denatures during thermal treatment when the temperature increases to 90°C-100°C but recovers native folding when the temperature decreases. Using this protein as a model, further studies show the maintenance of its bioactive functional properties after thermal stress, as demonstrated by differentiation induction of NIH/3T3 fibroblasts and human mesenchymal stem cells into myofibroblasts and chondrocytes, respectively. Molecular dynamic simulations revealed that although the protein's secondary structure is unstable under thermal stress, its conformation is partially stabilized by newly formed turns. Given the importance and/or prevalence of TGF-β1 in biological processes, potential therapeutics, and the human diet, our findings encourage consideration of its thermostability for biomedical applications and nutrition.

Figure 1

TGF-β1 unfolds at high temperatures but recovers when the temperature decreases. (A) CD spectra of TGF-β1 with the temperature increased from 25°C to 90°C. Arrows indicate peak shift directions. (B) CD spectra of TGF-β1 with the temperature decreased from 90°C to 25°C. Arrows indicate peak shift directions. (C) CD spectral values of TGF-β1 at 200 nm with temperature changes. (D) CD spectral values of TGF-β1 at 218 nm with temperature changes. To see this figure in color, go online.

Figure 2

CD spectra of TGF-β1 before and after boiling with or without TCEP. TCEP is a reduction reagent to break down disulfide bonds of the protein. (A) With TCEP. (B) Without TCEP. Measurements were taken at 25°C. Additional experimental replicates can be found in Fig. S1. To see this figure in color, go online.

Figure 3

Heated TGF-β1 maintains its ability to induce fibroblast differentiation to myofibroblasts after 48 h treatment. (A) Western blots of α-SMA expressed by NIH/3T3 cells after myofibroblast induction in the presence of untreated or thermally treated TGF-β1 at different incubation concentrations from 0.1 to 10 ng/mL. β-Actin served as the loading control. (B) Semi-quantitative analysis of two independent replicates of Western blots of α-SMA levels normalized to β-actin levels. Note that the error bars represent the standard deviation of two replicates. (C) Enzyme-linked immunosorbent assay of α-SMA expressed by NIH/3T3 cells after myofibroblast induction in the presence of untreated or thermally treated TGF-β1 at different boiling concentrations from 10 to 1,000 μg/mL (labeled as 10/100/1,000 boiled) and incubation concentrations from 0.1 to 10 ng/mL. The α-SMA level without induction was normalized to 100%. The error bars represent the standard deviation of four replicates. Groups within the same incubation concentration of TGF-β1 have no significant difference (p > 0.05; one-way ANOVA). Groups among different incubation concentrations exhibiting significant differences (p < 0.05; one-way ANOVA) have been labeled. To see this figure in color, go online.

Figure 4

GAG and DNA analysis of hMSC aggregates with untreated TGF-β1 or thermally treated TGF-β1 after 3 weeks of culture. (A) Results of biochemical assays. Asterisk (^∗) indicates significant difference analyzed by Student’s t-test (p < 0.05). The error bars represent the standard deviation of four replicates. (B) Histological images of tissue sections stained with Safranin O, a dye for GAG (red), and counterstained with Fast Green (green). To see this figure in color, go online.

Figure 5

Molecular dynamics simulations of TGF-β1 for 100 ns. (A) RMSD plots of TGF-β1 dimer and monomer chain A at 25°C and 100°C during molecular dynamics simulations. (B) Plots of secondary structure changes of TGF-β1 dimer at 100°C. Straight lines before 0 ns indicate the original numbers of each secondary structure prior to simulations. (C) Plots of secondary structure changes of TGF-β1 dimer at 25°C. Straight lines before 0 ns indicate the original numbers of each secondary structure prior to simulations. (D–G) Conformation changes of TGF-β1 simulated at 100°C (top) and 25°C (bottom). (D) Maps of secondary structures of TGF-β1 dimer before simulation and (E) heatmaps of TGF-β1 secondary structure changes simulated for 100 ns (color symbols: T, turn; E, β-sheet extended; B, β-bridge; H, α-helix; G, 3–10 helix; I, π-helix; C, coil). (F) Heatmaps of TGF-β1 RMSD changes simulated for 100 ns. (G) RMS fluctuations plots of amino acid residuals. Positions of amino acid residues (Table S2) in (D)–(G) are correlated. To see this figure in color, go online.