{'fraction_non_alphanumeric': 0.02666666666666667, 'fraction_numerical': 0.01611111111111111, 'mean_word_length': 5.210344827586207, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Metastable water clusters in the nonpolar cavities of the thermostable protein tetrabrachion.

Water expulsion from the protein core is a key step in protein folding. Nevertheless, unusually large water clusters confined into the nonpolar cavities have been observed in the X-ray crystal structures of tetrabrachion, a bacterial protein that is thermostable up to at least 403 K (130 degrees C). Here, we use molecular dynamics (MD) simulations to investigate the structure and thermodynamics of water filling the largest cavity of the right-handed coiled-coil stalk of tetrabrachion at 365 K (92 degrees C), the temperature of optimal bacterial growth, and at room temperature (298 K). Hydrogen-bonded water clusters of seven to nine water molecules are found to be thermodynamically stable in this cavity at both temperatures, confirming the X-ray studies. Stability, as measured by the transfer free energy of the optimal size cluster, decreases with increasing temperature. Water filling is thus driven by the energy of transfer and opposed by the transfer entropy, both depending only weakly on temperature. Our calculations suggest that cluster formation becomes unfavorable at approximately 384 K (110 degrees C), signaling the onset of drying just slightly above the temperature of optimal growth. "Drying" thus precedes protein denaturation. At room temperature, the second largest cavity in tetrabrachion accommodates a five water molecule cluster, as reported in the X-ray studies. However, the simulations show that at 365 K the cluster is unstable and breaks up. We suggest that the large hydrophobic cavities may act as binding sites for two proteases, possibly explaining the unusual thermostability of the resulting protease-stalk complexes (up to approximately 393 K, 120 degrees C).