Azido-derivatized amino acids are potentially useful, positionally resolved spectroscopic probes for studying the structural dynamics of proteins and macromolecules in solution. To this end a computational model for the vibrational modes of N − 3 based on accurate electronic structure calculations and a reproducing kernel Hilbert space representation of the potential energy surface for the internal degrees of freedom is developed. Fully dimensional quantum bound state calculations find the antisymmetric stretch vibration at 1974 cm −1 compared with 1986 cm −1 from experiment. This mode shifts by 64 cm −1 (from the frequency distribution) and 74 cm −1 (from the IRlineshape) to the blue, respectively, compared with 61 cm −1 from experiment for N − 3 in water. The decay time of the frequency fluctuation correlation function is 1.1 ps, in good agreement with experiment (1.2 to 1.3 ps) and the full width at half maximum of the asymmetric stretch in solution is 18.5 cm −1 compared with 25.2 cm −1 from experiment. A computationally more efficient analysis based on instantaneous normal modes is shown to provide comparable, albeit somewhat less quantitative results compared to solving the 3-dimensional Schrödinger equation for the fundamental vibrations.

The infrared spectroscopy and dynamics of −CO labels in wild type and mutant insulin monomer and dimer are characterized from molecular dynamics simulations using validated force fields. It is found that the spectroscopy of monomeric and dimeric forms in the region of the amide-I vibration differs for residues B24–B26 and D24–D26, which are involved in dimerization of the hormone. Also, the spectroscopic signatures change for mutations at position B24 from phenylalanine, which is conserved in many organisms and is known to play a central role in insulin aggregation, to alanine or glycine. Using three different methods to determine the frequency trajectories (solving the nuclear Schrödinger equation on an effective 1-dimensional potential energy curve, using instantaneous normal modes, and using parametrized frequency maps) leads to the same overall conclusions. The spectroscopic response of monomeric WT and mutant insulin differs from that of their respective dimers, and the spectroscopy of the two monomers in the dimer is also not identical. For the WT and F24A and F24G monomers, spectroscopic shifts are found to be ∼20 cm–1 for residues (B24–B26) located at the dimerization interface. Although the crystal structure of the dimer is that of a symmetric homodimer, dynamically the two monomers are not equivalent on the nanosecond time scale. Together with earlier work on the thermodynamic stability of the WT and the same mutants, it is concluded that combining computational and experimental infrared spectroscopy provides a potentially powerful way to characterize the aggregation state and dimerization energy of modified insulins.

Extensions and improvements of empirical force fields are discussed in view of applications to computational vibrational spectroscopy and reactive molecular dynamics simulations. Particular focus is on quantitative studies, which make contact with experiments and provide complementary information for a molecular-level understanding of processes in the gas phase and in solution. Methods range from including multipolar charge distributions to reproducing kernel Hilbert space approaches and machine learned energy functions based on neural networks.

The spectroscopic response of and structural dynamics around all azido-modified alanine residues (AlaN3) in lysozyme are characterized. It is found that AlaN3 is a positionally sensitive probe for the local dynamics, covering a frequency range of ∼15 cm−1 for the center frequency of the line shape. This is consistent with findings from selective replacements of amino acids in PDZ2, which reported a frequency span of ∼10 cm−1 for replacements of Val, Ala, or Glu by azidohomoalanine. For the frequency fluctuation correlation functions, the long-time decay constants τ2 range from ∼1 to ∼10 ps, which compares with experimentally measured correlation times of 3 ps. Attaching azide to alanine residues can yield dynamics that decays to zero on the few ps time scale (i.e., static component Δ0 ∼ 0 ps−1) or to a remaining, static contribution of ∼0.5 ps−1 (corresponding to 2.5 cm−1), depending on the local environment on the 10 ps time scale. The magnitude of the static component correlates qualitatively with the degree of hydration of the spectroscopic probe. Although attaching azide to alanine residues is found to be structurally minimally invasive with respect to the overall protein structure, analysis of the local hydrophobicity indicates that the hydration around the modification site differs for modified and unmodified alanine residues, respectively.

The spectroscopy and structural dynamics of a deep eutectic mixture (KSCN/acetamide) with varying water content is investigated from 2D IR (with the C–N stretch vibration of the SCN– anions as the reporter) and THz spectroscopy. Molecular dynamics simulations correctly describe the nontrivial dependence of both spectroscopic signatures depending on water content. For the 2D IR spectra, the MD simulations relate the steep increase in the cross-relaxation rate at high water content to the parallel alignment of packed SCN– anions. Conversely, the nonlinear increase of the THz absorption with increasing water content is mainly attributed to the formation of larger water clusters. The results demonstrate that a combination of structure-sensitive spectroscopies and molecular dynamics simulations provides molecular-level insights into the emergence of heterogeneity of such mixtures by modulating their composition.