Water, protons, and ions play a central role in the stability, dynamics, and function of biomolecules. 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 biological proton pumps and enzymes. We have made advances in several 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. By using molecular dynamics simulations we could characterize the energetics and kinetics of ion conduction through the GLIC ion channel (1-3). Importantly, we could demonstrate that a recent crystal structure of GLIC corresponds to a functionally closed state, with the calculated ion conductance in agreement with the measured conductance (1). We could also shed light on the mechanism employed by GLIC and possibly other channels to gate the ion flow. Remarkably, we found that conductance of ions through GLIC in its functionally closed state is blocked by removal of water from a 15 Angstrom hydrophobic constriction (3), in response to a conformational change that tightens the pore. Whereas ions can still pass relatively easily through a pre-hydrated constriction, the high energetic cost of hydration effectively blocks ion passage. This amounts, arguably, to the first quantitative demonstration of a direct functional role of molecular drying. Biological proton pumps Complex I and cytochrome c oxidase. Aerobic life is based on a molecular machinery that utilizes oxygen as a terminal electron sink. Complex I is a key entry point into the respiratory chain of mitochondria and several bacteria functions that itself functions as a redox-driven proton pump. In collaboration with Prof. Wikstrom (University of Helsinki) we re-examined the stoichiometry of proton translocation, as a factor essential for a proper understanding of this key enzyme (5). On the basis of the recent structure and our stoichiometric analysis, we developed a rough mechanistic model involving concerted proton translocation in the three homologous and tightly packed antiporter-like subunits of the trans-membrane domain (5). We also studied the membrane-bound cytochrome c oxidase (CcO), which catalyzes the reduction of oxygen to water in mitochondria and many bacteria. The energy released in this reaction is conserved by pumping protons across the mitochondrial or bacterial membrane, creating an electrochemical proton gradient that drives the production of ATP. In collaboration with Dr. Kim (Naval Research Lab, Washington, DC), we developed detailed kinetic models of the redox-coupled proton pump in CcO. These models have allowed us not only to explain how redox chemistry can be harnessed to charge up the inner mitochondrial membrane to power aerobic life, but also how modifications in the machinery affect its efficiency (6). Interior hydration of proteins. We have studied the energetics of water forming one-dimensional wires inside a nonpolar channel (4), as seen proton and water conducting proteins. We could show that the entropy associated with the formation of such water chains is negative, i.e., unfavorable. As a result the water chains are predicted to be unstable at elevated temperatures. Proton-coupled electron transfer direct and water mediated. Proton-coupled electron transfer (PCET) reactions are essential to many biological processes, ranging from photosynthesis and energy transduction in mitochondria (5) to enzymatic catalysis (7). We performed quantum chemical calculations to study the direct and water-mediated PCET between two stacked tyrosines. This system mimics a key step in the catalytic reaction of class Ia ribonucleotide reductases. We found that the pi-stacking of the tyrosine dimer results in strong electronic coupling and effective adiabatic PCET. We also showed that water participation in the PCET can be identified perturbatively. 1. F. Zhu, G. Hummer, Theory and simulation of ion conduction in the pentameric GLIC channel, J. Chem. Theory Comput., in press (2012). http://dx.doi.org/10.1021/ct2009279 2. F. Zhu, G. Hummer, Drying transition in the hydrophobic gate of the GLIC channel blocks ion conduction, Biophys. J. 103, 219-227 (2012). 3. F. Zhu, G. Hummer, Convergence and error estimation in free energy calculations using the weighted histogram analysis method, J. Comp. Chem. 33, 453-465 (2012). 4. Waghe, J. C. Rasaiah, G. Hummer, Entropy of single-file water in (6, 6) carbon nanotubes, J. Chem. Phys. 137, 044709 (2012). 5. M. Wikstrm, G. Hummer, Stoichiometry of proton translocation by respiratory Complex I and its mechanistic implications, Proc. Natl. Acad. Sci. USA 109, 4431-4436 (2012). 6. Y. C. Kim, G. Hummer, Proton-pumping mechanism of cytochrome c oxidase: A kinetic master-equation approach, Biochim. Biophys. Acta-Bioenergetics 1817, 526-536 (2012). 7. V. R. I. Kaila, G. Hummer, Energetics of direct and water-mediated proton-coupled electron transfer, J. Am. Chem. Soc. Communication 133, 1904019043 (2011).

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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
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) Theory and simulation of ion conduction in the pentameric GLIC channel. J Chem Theory Comput 8:3759-3768
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
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

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