Water, protons, and ions play a central role in the stability, dynamics, and function of biomolecules, and are also an important factor in the binding of drug molecules (1). Through the hydrophobic effect and hydrogen bond interactions, water is a major factor in the folding of proteins. In many enzymes, it participates directly in the catalytic function. In particular, water in the protein interior often mediates the transfer of protons between the solvent medium and the active site. Such water, often confined into relatively nonpolar pores and cavities of nanoscopic dimensions, exhibits highly unusual properties, such as high water mobility, high proton conductivity, or sharp transitions between filled and empty states. Proteins exploit these unusual properties of confined water in their biological function, e.g., to ensure rapid water flow in aquaporins, or to gate proton flow in proton pumps and enzymes. Water is a key factor in the binding and recognition process (2), and determines the friction at the nanoscale (3). We have made a number of advances in areas where water, protons, and ions are connected to protein function. Ion channel gating. Nerve signaling in humans and chemical sensing in bacteria both rely on the controlled opening and closing of the ion-conducting pore in pentameric ligand-gated ion channels. With the help of a multiscale simulation approach that combined a low-resolution elastic model with atomistic molecular dynamics simulations, we studied the opening and closing of the pore in GLIC, a prokaryotic channel (4). We found that the pore closes in an iris-like fashion, with the pore-lining helices collectively tilting with respect to the membrane normal. This motion induces a cooperative drying transition of the channel pore, in which the water rapidly exits from its central nonpolar region. The mechanical work of opening the pore is performed primarily on the M2-M3 loop. Strong interactions of this short and conserved loop with the extracellular domain are therefore crucial to couple ligand binding to channel opening. Interior hydration of proteins. We used simulations to resolve a long-standing question in protein-water interactions: whether the nonpolar cavity in the protein interleukin-1βis filled by water or empty (5). With the help of molecular dynamics simulations, we studied the thermodynamics of filling the central nonpolar cavity and the four polar cavities of interleukin-1β. We found that water in the central nonpolar cavity is thermodynamically unstable, independent of simulation force field and water model. The apparent reason is the relatively small size of the cavity, with a volume less than 80 cubic Angstrom. Our results are consistent with the most recent X-ray crystallographic and simulation studies, but disagree with an earlier interpretation of nuclear magnetic resonance (NMR) experiments probing protein-water interactions. To resolve this apparent discrepancy we showed that the measured nuclear Overhauser effects can, in all likelihood, be attributed to interactions with buried and surface water molecules near the cavity. Our study thus resolves the long-standing controversy concerning the presence of water in interleukin-1β. Single-file water as a proton wire. With a quantum mechanical description we studied the transfer of protons along an ordered chain of water molecules (6). Such water chains have highly unusual properties, including a strong dipolar order (7). We found that for short water chains with four water molecules, the proton transfer reaction is semi-concerted. We also showed that the barrier of the pT reaction depends linearly on the proton affinity of the donor but is nearly independent of the proton affinity of the acceptor, corresponding to Bronsted slopes of one and zero, respectively. These simulations provide a detailed picture of an essential step in many biochemical reactions. 1. G. Hummer, Molecular binding: under waters influence, Nature Chemistry 2, 906-907 (2010). 2. J. Mittal, G. Hummer, Interfacial thermodynamics of confined water near molecularly rough surfaces, Faraday Discuss. 146, 341-352 (2010). 3. A. Kalra, S. Garde, G. Hummer, Lubrication by molecularly thin water films confined between nanostructured membranes, Eur. Phys. J. Special Topics 189, 147-154 (2010). 4. F. Zhu, G. Hummer, Pore opening and closing of a pentameric ligand-gated ion channel, Proc. Natl. Acad. Sci. USA 107, 19814-19819 (2010). 5. H. Yin, G. Feng, G. M. Clore, G. Hummer, J. C. Rasaiah, Water in the polar and nonpolar cavities of the protein interleukin-1β, J. Phys. Chem. B 114, 16290-16297 (2010) 6. V. R. I. Kaila, G. Hummer, Energetics and dynamics of proton transfer reactions along short water wires, Phys. Chem. Chem. Phys. 13, 13207-13215 (2011). 7. J. Kfinger, G. Hummer, C. Dellago, Single-file water in nanopores, Phys. Chem. Chem. Phys. 13, 15403 - 15417 (2011).

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Waghe, Aparna; Rasaiah, Jayendran C; Hummer, Gerhard (2012) Entropy of single-file water in (6,6) carbon nanotubes. J Chem Phys 137:044709
Zhu, Fangqiang; Hummer, Gerhard (2012) Drying transition in the hydrophobic gate of the GLIC channel blocks ion conduction. Biophys J 103:219-27
Zhu, Fangqiang; Hummer, Gerhard (2012) Convergence and error estimation in free energy calculations using the weighted histogram analysis method. J Comput Chem 33:453-65
Kim, Young C; Hummer, Gerhard (2012) Proton-pumping mechanism of cytochrome c oxidase: a kinetic master-equation approach. Biochim Biophys Acta 1817:526-36
Wikstrom, Marten; Hummer, Gerhard (2012) Stoichiometry of proton translocation by respiratory complex I and its mechanistic implications. Proc Natl Acad Sci U S A 109:4431-6
Kaila, Ville R I; Hummer, Gerhard (2011) Energetics of direct and water-mediated proton-coupled electron transfer. J Am Chem Soc 133:19040-3
Köfinger, Jürgen; Hummer, Gerhard; Dellago, Christoph (2011) Single-file water in nanopores. Phys Chem Chem Phys 13:15403-17
Kaila, Ville R I; Hummer, Gerhard (2011) Energetics and dynamics of proton transfer reactions along short water wires. Phys Chem Chem Phys 13:13207-15
Hummer, Gerhard (2010) Molecular binding: Under water's influence. Nat Chem 2:906-7
Yin, Hao; Feng, Guogang; Clore, G Marius et al. (2010) Water in the polar and nonpolar cavities of the protein interleukin-1?. J Phys Chem B 114:16290-7

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