Fig 1.
Left, the positions of the catalytic triad, tryptophan residues and structure of the S1 pocket of trypsin consists of residues 184–195 (Loop 1), 213–228 (Loop 2). Loop 3 (169–175) is not shown. Right, the native structure of trypsin (PDB ID: 1FNI).
Fig 2.
(a) and (b) the first and the last snapshots of trajectory in system 1, respectively. (c) and (d) the first and last snapshots of trajectory in system 2, respectively.
Fig 3.
(a) Fluorescence spectra of the COOH-f-MWCNTs-pTry system. Conditions: λex = 280 nm, pH 8.0; C trypsin 2.0 × 10−5 M, CCNTs are in the total volume of the reaction mixture; pH 8.0, T = 298 K. (b) Emission spectra of COOH-f-MWCNTs systems alone. (c) The plot of fluorescence quenching for trypsin with the concentration of COOH-f-MWCNTs. (d) The logarithmic plot of trypsin with the concentration of COOH-f-MWCNTs. Data are shown as mean values ± S.D.
Fig 4.
(a) UV–visible absorption spectra of trypsin. C trypsin = 2.0 × 10−5 M, CCNTs are in the total volume of the reaction mixture; pH 8.0, T = 298 K. (b) CD spectra of trypsin and COOH-f-MWCNTs-pTry mixtures. C trypsin = 1 × 10−4 M. CCNTs are in the total volume of the reaction mixture; pH 8.0, T = 298 K. Data are shown as mean values ± S.D.
Fig 5.
(a) Effects of COOH-f-MWCNTs on the activity of trypsin. (b) The thermostability of trypsin in the presence of COOH-f-MWCNTs. pH 8.0, T = 330 K: C trypsin (control) = 100 μg/ml. CCNTs are in the total volume of the reaction mixture; pH 8.0, T = 298 K. Data are shown as mean values ± S.D.
Table 1.
The influence of COOH-f-MWCNTs on the secondary structure of trypsin.
Table 2.
Effect of COOH-f-MWCNTs on the kinetic parameters of the porcine trypsin.
The values were calculated using GraphPad Prism.
Table 3.
Inactivation rate constant (kin) and half-life of the trypsin in the absence and presence of COOH-f-MWCNTs at T = 330 K.
Fig 6.
(a) Center of mass (COM) distances between the enzyme and COOH-f-DWCNT as a function of time (b) The RMSD of C-α of trypsin during the simulation time.
Fig 7.
Time evolution of the secondary structure of the enzyme during MD simulations generated by DSSP for (a) the enzyme in water (as control), (b) system 1, and (c) system 2. The cartoon model of the first and the last snapshots of MD of the enzyme structure are shown in left and right respectively. Changes of secondary structure of several residues are shown, for instance. The alpha helix, 310 helix, beta sheet, turns, and coil structures are colored in violet, cyan, magenta, orange, and ice blue, respectively. For the DSSP analysis of the enzyme in system 3 and system 4, refer to the supplementary information.
Fig 8.
MD simulations indicate interaction between COOH-f-DWCNT and S1 pocket of the enzyme.
(a) Minimum distance between Loop 1 and Loop 2 of S1 pocket with COOH-f-DWCNT in system 1 over the simulation time (for analysis of minimum distance between Loops with COOH-f-DWCNT in system 3, refer to the supplementary information). (b) The number of hydrogen bonds between S1 pocket and COOH groups of COOH-f-DWCNT in system 1 as function of time. (c) Local snapshots from hydrogen binding interaction by (I) Gln-221, (II) Lys-222, and (III) Lys-224 and π-π stacking interaction by (IV) Tyr-217.
Table 4.
The binding energy (ΔGbinding) of S1 pocket residues (kJ.mol-1) in system1.
Fig 9.
The snapshot of MD simulation outcomes shows the preferred binding sites on enzyme for COOH-f-DWCNT.
π-π stacking interactions (a) Tyr 151 and Tyr 217 (system 1) (b) Tyr 29 (system 2). π–cation stacking interactions (c, d) Lys 60 and Arg 62 (system 1) (e, f, g) Arg 117, Arg 125 and Lys 159 (sysytem2). (h) Energetic components of COOH-f-DWCNT-pTry complex. vdW is van der Waals energy, SASA is non-polar energy, Elec is electrostatic energy, Pols is polar solvation energy, Npb is non-polar binding energy and Pb is polar binding energy.
Fig 10.
Adsorption process of the enzyme on the surface of COOH-f-DWCNT.
(a) to (d) The minimum distance between residues and COOH-f-DWCNT in system 1, system 2, system 3 and system 4, respectively. (e) to (h) the energy residues with value of binding energy < -2 kJ.mol−1 in system 1, system 2, system 3 and system 4, respectively.