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It remains unclear how the abundant charged residues in proteins from hyperthermophiles contribute to the stabilization of proteins. Previously, based on molecular dynamics (MD) simulations, we proposed that these charged residues decrease the entropic effect by forming salt bridges in the denatured state under physiological conditions (Yutani et al ., Sci. Rep . 8, 7613 (2018)). Because the quality of MD results is strongly dependent on the force fields used, in this study we performed the MD simulations using a different force field (AMBER99SB) along with the one we used before (Gromos43a1), at the same temperatures examined previously as well as at higher temperatures. In these experiments, we used the same ionic mutant (Ec0VV6) of CutA1 from Escherichia coli as in the previous study. In MD simulations at 300 K, Lys87 and Arg88 in the loop region of Ec0VV6 formed salt bridges with different favorable pairs in different force fields. Furthermore, the helical content and radius of gyration differed slightly between two force fields. However, at a higher temperature (600 K), the average numbers of salt bridges for the six substituted residues of Ec0VV6 were 0.87 per residue for Gromos43a1 and 0.88 for AMBER99SB in 400-ns MD simulation, indicating that the values were similar despite the use of different force fields. These observations suggest that the charged residues in Ec0VV6 can form a considerable number of salt bridges, even in the denatured state with drastic fluctuation at 600 K. These results corroborate our previous proposal., Competing Interests: Conflict of Interest The authors declare no competing interests., (2019 © The Biophysical Society of Japan.)

Terahertz time-domain spectra (THz-TDS) were investigated using the results of molecular dynamics (MD) simulations of Staphylococcal nuclease at two hydration states in the temperature range between 100 and 300 K. The temperature dependence of THz-TDS was found to differ significantly from that of the incoherent neutron scattering spectra (INSS) calculated from the same MD simulation results. We further examined contributions of the mutual and auto-correlations of the atomic fluctuations to THz-TDS and found that the negative value of the former contribution nearly canceled out the positive value of the latter, resulting in a monotonic increase of the reduced absorption cross section. Because of this cancellation, no distinct broad peak was observed in the absorption lineshape function of THz-TDS, whereas the protein boson peak was observed in INSS. The contribution of water molecules to THz-TDS was extremely large for the hydrated protein at temperatures above 200 K, in which large-amplitude motions of water were excited. The combination of THz-TDS, INSS and MD simulations has the potential to extract function-relevant protein dynamics occurring on the picosecond to nanosecond timescale., Competing Interests: Conflict of Interest Y. J. and A. K. declare that they have no conflict of interest., (2019 © The Biophysical Society of Japan.)