| Abstract
| - Water expulsion from the protein core is a key step in protein folding. Nevertheless, unusuallylarge water clusters confined into the nonpolar cavities have been observed in the X-ray crystal structuresof tetrabrachion, a bacterial protein that is thermostable up to at least 403 K (130 °C). Here, we use moleculardynamics (MD) simulations to investigate the structure and thermodynamics of water filling the largestcavity of the right-handed coiled-coil stalk of tetrabrachion at 365 K (92 °C), the temperature of optimalbacterial growth, and at room temperature (298 K). Hydrogen-bonded water clusters of seven to nine watermolecules are found to be thermodynamically stable in this cavity at both temperatures, confirming theX-ray studies. Stability, as measured by the transfer free energy of the optimal size cluster, decreases withincreasing temperature. Water filling is thus driven by the energy of transfer and opposed by the transferentropy, both depending only weakly on temperature. Our calculations suggest that cluster formationbecomes unfavorable at ∼384 K (110 °C), signaling the onset of drying just slightly above the temperatureof optimal growth. “Drying” thus precedes protein denaturation. At room temperature, the second largestcavity 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 thelarge hydrophobic cavities may act as binding sites for two proteases, possibly explaining the unusualthermostability of the resulting protease-stalk complexes (up to ∼393 K, 120 °C).
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