Multiscale theoretical study of infrared spectroscopy for the thermal denaturation process of aprotinin

The stability of protein secondary structure is the basis for the realization of biological functions, and temperature induces conformational changes in proteins by disrupting the equilibrium of the internal hydrogen bond network. This study systematically elucidates the temperature-induced unfolding mechanism of aprotinin based on one-dimensional infrared spectroscopy (1D IR) and two-dimensional infrared spectroscopy (2D IR) simulated by molecular dynamics (MD) simulations. The simulations showed that the characteristic absorption peaks of the amide I band in the IR spectra of the aprotinin were blueshifted and weakened with increasing temperature, indicating that the hydrogen bond breaking leads to the secondary structure transformation of the aprotinin. The 2D IR simulations of aprotinin reveal that when the temperature rose to 333 K, the vibrational coupling peak of the β-sheet at (1625.4 cm^-1, 1653.4 cm^-1) disappears. At 353 K, new coupling peaks corresponding to random coil and β-turn appear at (1639.7 cm^-1, 1672.2 cm^-1), and these signals disappear at 373 K, indicating complete unfolding of aprotinin. During the heating process, the 2D IR spectral signal of the aprotinin shifted from excited-state absorption (ESA) to ground-state bleaching (GSB), reflecting the energy transfer process within the protein, and its dynamics process and spectral broadening rate were greatly affected by temperature. The unfolding pathway of aprotinin was again obtained by MD simulation and analyzed in comparison with infrared spectroscopy, and the unfolding behavior of the aprotinin was resolved at the atomic level.

In this work, the MD simulation of aprotinin was carried out by GROMACS, using the OPLS/AA force field and SPC/E water model, with a simulation duration of 500 ns and a time step of 2 fs. After the simulation, the C = O stretching vibrations of peptide bonds were extracted from the trajectory files to calculate the autocorrelation function and third-order nonlinear response function, which were Fourier transformed to obtain the 1D and 2D IR spectra of aprotinin. The DSSP program in GROMACS was used to quantitatively analyze the secondary structure content of aprotinin at various temperatures, and the conformational changes under different temperature conditions were visually analyzed by using VMD software.