Ere are interactions between the transitions of W5 and ??W16 (spaced at 5.4 A); W97 and W245 (8.0 A); W192 and W209 ???(10.4 A); W123 and Y128 (10.1 A); W192 and Y191 (8.6 A); and ?Y194 and W209 (3.9 A). Nevertheless, it is clear that tryptophans participate in several coupling interactions: the one electron mixing type of interactions tend to exhibit higher interaction energies with at least one order of magnitude higher than the coupled oscillator type ones (Table 1). The results are in agreement with MedChemExpress BIBS39 earlier studies on class A b-lactamases, which revealed that the one electron effect is the prefered mechanism by which tryptophans generate the strongest contributions to the near-UV CD spectra [20,32,33].Influence of Conformational Flexibility on the Calculated CD Spectra of the Wild- Type HCAIIProteins are characterized by intrinsic conformational flexibility which might influence their structural properties and functions [34,35] and MD is one of the most widely utilized techniques forexploration of their conformational dynamics [36]. Since CD spectra are a consequence of the mutual orientation and distances of the protein chromophores within the protein structure, conformational flexibility would exercise an influence on the chiroptical properties of proteins, e.g. on the quality of the predicted CD spectra and the nature of the underlying mechanisms. To explore this important issue 20 ns MD simulations of the wild-type enzyme were performed and the CD spectra using 40 random structures (snapshots) along the MD trajectory were calculated. The averaged spectrum over the calculated MD snapshots provides almost a two-fold better agreement to the experimental one for the main near-UV spectral feature (the SMER28 custom synthesis minimum at 270 nm in the experimental spectrum and 263 nm in the calculated one), in contrast to the calculated spectrum based on the X-ray crystal structure alone (Figure 2A, in red). In order to facilitate the comparison, we presented also scaled computed spectra which were received through red shifting of the original ones by 6 nm (presented in Figure 2A with dashed blue and dashed red lines, respectively for the crystal structure and MDaveraged scaled spectra). Up to 267 nm (275 nm for the scaled spectra) the MD averaged calculations provide better agreement to the experimental one, and above this wavelength the calculations based on the crystal structure show closer magnitudes to the experiment. Above 280 nm (287 nm for the scaling corrected spectra) the MD-based spectrum shows slightly positive sign (in contrast to the experiment and the calculations based on theConformational Effects on the Circular Dichroismcrystal structure only). This could be due to interactions in nonfavorable protein 15900046 conformations. Its intensity, however, is relatively small and would not diminish the better agreement achieved for the main spectral feature. In the far-UV region, the averaged spectra calculated over the MD snapshots provide some improvement to the predictions of the CD spectral magnitudes as well, however the results are still far from being in a good agreement with the experimental data (Fig. 2B, with semiempirical monopoles in yellow, and with ab initio ones in red).Mechanistic Effects of the Conformational ChangesCombining CD calculations and MD enables exploration of the influence of the protein conformational flexibility on the mechanisms of generation of rotational strengths and chromophore interactions, thus facilitating a deeper insi.Ere are interactions between the transitions of W5 and ??W16 (spaced at 5.4 A); W97 and W245 (8.0 A); W192 and W209 ???(10.4 A); W123 and Y128 (10.1 A); W192 and Y191 (8.6 A); and ?Y194 and W209 (3.9 A). Nevertheless, it is clear that tryptophans participate in several coupling interactions: the one electron mixing type of interactions tend to exhibit higher interaction energies with at least one order of magnitude higher than the coupled oscillator type ones (Table 1). The results are in agreement with earlier studies on class A b-lactamases, which revealed that the one electron effect is the prefered mechanism by which tryptophans generate the strongest contributions to the near-UV CD spectra [20,32,33].Influence of Conformational Flexibility on the Calculated CD Spectra of the Wild- Type HCAIIProteins are characterized by intrinsic conformational flexibility which might influence their structural properties and functions [34,35] and MD is one of the most widely utilized techniques forexploration of their conformational dynamics [36]. Since CD spectra are a consequence of the mutual orientation and distances of the protein chromophores within the protein structure, conformational flexibility would exercise an influence on the chiroptical properties of proteins, e.g. on the quality of the predicted CD spectra and the nature of the underlying mechanisms. To explore this important issue 20 ns MD simulations of the wild-type enzyme were performed and the CD spectra using 40 random structures (snapshots) along the MD trajectory were calculated. The averaged spectrum over the calculated MD snapshots provides almost a two-fold better agreement to the experimental one for the main near-UV spectral feature (the minimum at 270 nm in the experimental spectrum and 263 nm in the calculated one), in contrast to the calculated spectrum based on the X-ray crystal structure alone (Figure 2A, in red). In order to facilitate the comparison, we presented also scaled computed spectra which were received through red shifting of the original ones by 6 nm (presented in Figure 2A with dashed blue and dashed red lines, respectively for the crystal structure and MDaveraged scaled spectra). Up to 267 nm (275 nm for the scaled spectra) the MD averaged calculations provide better agreement to the experimental one, and above this wavelength the calculations based on the crystal structure show closer magnitudes to the experiment. Above 280 nm (287 nm for the scaling corrected spectra) the MD-based spectrum shows slightly positive sign (in contrast to the experiment and the calculations based on theConformational Effects on the Circular Dichroismcrystal structure only). This could be due to interactions in nonfavorable protein 15900046 conformations. Its intensity, however, is relatively small and would not diminish the better agreement achieved for the main spectral feature. In the far-UV region, the averaged spectra calculated over the MD snapshots provide some improvement to the predictions of the CD spectral magnitudes as well, however the results are still far from being in a good agreement with the experimental data (Fig. 2B, with semiempirical monopoles in yellow, and with ab initio ones in red).Mechanistic Effects of the Conformational ChangesCombining CD calculations and MD enables exploration of the influence of the protein conformational flexibility on the mechanisms of generation of rotational strengths and chromophore interactions, thus facilitating a deeper insi.
