Fig 1.
Thermal folding-unfolding of MTH1880.
(A) Data were acquired at 25°C (black triangle), 45°C (red square), 65°C (blue circle), 75°C (yellow square), 85°C (red triangle), 95°C (green square), and 105°C (red circle). Protein concentration was ~25 μM in a cell of 0.1 mm path length. (B) The fraction of unfolding extracted from far-UV CD spectra at 222 nm with a constant heating rate of 10°C/h as a function of temperature was plotted and fit by a sigmoidal curve. The transition mid-temperature (Tm) of MTH1880 was 76 ± 0.5°C. (C) Results from molecular dynamics (MD) simulations. (Left panel) Radius of gyrations (Rg) of MTH1880 as a function of time at temperatures from 300 K to 525 K. (Middle panel) Rg averaged over 400,000 snapshots in the time window from 100 ns to 500 ns. The error bar denotes one standard deviation. (Right panel) The probability distribution of Rg is plotted for each temperature and results in sharp distributions at low temperatures and broad distributions at high temperatures. (D) (Left panel) Atomic fluctuation (RMSF) of MTH1880 by residue in the same time window and same temperatures as (C). (Right panel) RMSF averaged over 88 residues. (C, D) This data were acquired at 300K(red filled circle), 325K(green filled triangle), 350K(blue filled triangle), 375K(pink filled square), 400K(cyan cross), 425K(black cross), 450K(red circle), 475K(green triangle), 500K(blue triangle), and 525K(pink square). (E) Secondary structures of MTH1880 are shown with residue numbers. A ladder diagram displays residue-residue pairwise contacts denoted by a semicircle line with the distance cut-off 6.5A to emphasize the major topology of pairwise residue-residue interactions based on three-dimensional structure of MTH1880. (F) Extended Munoz-Eaton (ME) model. Data were acquired at 0.7Tm(black reverse triangle), 0.8Tm(pink square), 0.9Tm(green cross), 1.0Tm(red circle), 1.1Tm(cyan cross), 1.2Tm(blue square), and 1.3Tm(black triangle). (Left panel) Free energy (ΔG) landscape of MTH1880 as a function of the reaction coordinate, where M = 0 denotes the fully denatured structure and M = 1 denotes the native structure. The free energy of folding ΔΔGD-N (ΔGD—ΔGN, right top panel) and the fraction of unfolded protein as a function of the reduced temperature (T/Tm), where Tm is the transition mid-temperature (right bottom panel). At T = Tm, ΔΔGD-N = 0, and the fraction of unfolding is 0.5. (G) Correlation matrix of MTH1880 at 300 K is calculated from 400,000 snapshots in the same time window as (C). The secondary structure of MTH1880, in the N to C direction, are depicted next to the axes. (H) Correlation matrix of MTH1880 at (Top left to right) 325K, 350K, 375K, (Middle left to right) 400K, 425K, 450K, (Bottom left to right) 475K, 500K and 525K.
Fig 2.
Unfolding of MTH1880 in the presence of urea.
(A) Data were acquired at 0 M (black triangle), 2 M (blue circle), 3 M (yellow diamond), (red square), 4.5 M (green circle), 5 M (magenta square), 5.5 M (dark green triangle), 6 M (red circle), 6.5 M (magenta filled diamond), 7 M (green square), 7.5 M (green triangle), 8 M (blue triangle), 8.5 M (khaki diamond) and 9 M (yellow circle) urea. (B) The fraction of unfolded protein extracted from far-UV CD spectra (25 μM) at 222 nm as a function of urea concentration was plotted and fit by a sigmoidal curve. The transition mid-concentration of urea (Cm) is 6.10 ± 0.15 M. (C) Fluorescence spectra acquired for urea concentrations ranging from 0 to 9.0 M. Data were acquired for 0 M (red line), 2M (fluorescent green line), 3 M (purple line), 4 M (blue), 6 M (brown), 6.5 M (magenta), 7 M (sky-blue), 7.5 M (khaki), 8 M (yellow), 8.5 M (light sky-blue) and 9 M (orange). (D) The fraction of unfolded protein extracted from fluorescence-emission spectra (25 μM) at 308 nm as a function of urea concentration was plotted and fit by a sigmoidal curve. The transition mid-concentration of urea is 6.00± 0.05 M. (E) Results from MD simulations. (Left panel) Rg of MTH1880 as a function of time, with urea concentrations ranging from 0 to 8 M at 300 K. (Middle panel) Rg averaged over 300,000 snapshots taken in the time window of 200 to 500 ns. The error bar denotes one standard deviation. (Right panel) The probability distribution of Rg is plotted for each urea concentration, and results in sharp distributions at low temperatures and broad distributions at high temperatures. (F) (Left panel) RMSF of MTH1880 by residue in the same time window, same temperatures and same urea concentration as (E). (Right panel) RMSF averaged over 88 residues. (E, F) This data were acquired for urea 0M(red filled circle), 1M(green filled triangle), 2M(blue filled triangle), 3M(red square), 4M(cyan cross), 5M(black cross), 6M(red circle), 7M(green triangle), and 8M(blue triangle). (G) Results from employing the extend ME model. Data were acquired for urea 0M(red circle), 1M(green cross), 2M(blue square), 3M(red cross), 4M(cyan square), 5M(black triangle), 6M(grey triangle), 7M(purple star), and 8M(blue filled circle). (Left panel) Free energy landscape of MTH1880 at 1.0Tm for each urea concentration as a function of M. (Middle top panel). The free energy of folding ΔΔGD-N (middle bottom panel) and the fraction of unfolded protein for each urea concentration, as a function of T/Tm. At T = Tm, ΔΔGD-N = 0, and the fraction of unfolding is 0.5. (Right top panel) The free energy of folding ΔΔGD-N (right bottom panel) and the fraction of unfolded protein for the temperature interval of 0.1Tm to 1.2Tm, as a function of urea concentration. At the transition mid-concentration of urea, ΔΔGD-N = 0, and the fraction of unfolding is 0.5. (H) Correlation matrix of MTH1880 is calculated from 300,000 snapshots in the same time window as (E) with urea concentration (Top left to right) 0M, 1M, 2M, (Middle left to right) 3M, 4M, 5M, (Bottom left to right) 6M, 7M and 8M.
Fig 3.
GdnHCl denaturation monitored by circular dichroism and fluorescence spectroscopy.
(A) Data were acquired at 0 M (black triangle), 1 M (red square), 2 M (blue circle), 3 M (hkaki filled diamond),4.5 M (red triangle), 5 M (green square), 6 M (red circle), 7 M (hkaki diamond), and 8 M (cyan cross) urea. (B) The fraction of unfolding extracted from far-UV CD spectra (25 μM) at 222nm as a function of GdnHCl concentration was plotted and fit by a sigmoidal curve. The transition mid-concentration of GdnHCl (Cm) is 3.95 ± 0.1 M. (C) Fluorescence-emission spectra for different GdnHCl concentrations ranging from 0 to 6.0 M. (D) The fraction of unfolding extracted from fluorescence-emission spectra (25 μM) at 308 nm, as a function of urea concentration, was plotted and fit by a sigmoidal curve. The transition mid-concentration of urea is 4.1 ± 0.05 M.
Fig 4.
(A) Superimposition of Far-UV CD spectra for the native state (black triangle) and refolded state (red circle), which was reached by lowering to 25°C from 105°C. (B) Far-UV CD spectra for the native state (black triangle) and refolded state (red circle), which was reached by diluting GdnHCl from 6 to 0 M. (C) Size-exclusion FPLC profiles of MTH1880 as a function of urea and GdnHCl concentration of 0 M (red line), 2 M (blue line), 3 M (green line), 4 M (yellow line), 5 M (brown line), 6 M (purple line), 8 M (black line), and 0 M (refolded, red dotted line). (D) Overlay of the two-dimensional 1H-15N HSQC spectra for MTH1880 are shown in both the native state (black) and refolded state (red) at 36°C.
Fig 5.
Residue-specific characteristics of MTH1880 unfolding.
(A) Chemical shift perturbation (CSP) analysis to detect residues susceptible to urea denaturation. The average chemical-shift changes were calculated using the following formula: ΔδAV = [(Δδ1H)2+(Δδ15N/5)2]1/2, where Δδ AV, Δδ 1H, and Δδ 15N are the average, proton, and 15N chemical-shift changes, respectively. (B) Structure of the MTH1880 represented by a ribbon diagram. K13-D36 and K13-D38 form salt bridges. Dashed lines indicate the salt bridges. K13-D36 and K13-D38 salt bridges contributed to the stability of the folding structure of MTH1880. Red and blue atoms mean oxygen and nitrogen, respectively. Hydrophobic core is formed by side-chain connectivity of hydrophobic residues. It is represented by sphere and stick, respectively. (C) Thermal-induced denaturation curves of wild type MTH1880 and mutants. The fraction of unfolding extracted from far-UV CD spectra at 222 nm with a constant heating rate of 10°C/h and 25μM protein concentrations. MTH1880 wt (filled circle), mutants in the salt bridge; K13A (triangle), D36A (square), D38A (circle) and mutants in the hydrophobic pocket; V23A (diamond), V53A(filled square). (D) Thermal stabilities were investigated for MTH1880 mutants. Tm values of mutant proteins are indicated as black bars.