Lateral proton migration along membranes

Transmembrane proton concentration gradients drive the mitochondrial ATP-synthase or facilitate transport across the bacterial translocation channel. These gradients differ from their respective counterparts in the bulk because an energy barrier, G, delays proton surface-to-bulk transfer, thereby enabling proton migration towards the respective molecular machine along the membrane surface. This interfacial proton migration is also likely to be important for a variety of other processes, like proton coupled uptake of nutrients, proton delivery to active centers of proteins, and protein folding. According to conflicting theories, proton surface diffusion occurs (i) totally decoupled from bulk or (ii) under conditions of rapid equilibrium between bulk and surface protons. Here we aim to distinguish between both theories. Therefore, we directly observe surface proton diffusion by releasing the protons either instantaneously or gradually at one spot on planar membranes and by detecting their arrival at a distant spot. By identifying the conditions under which transmembrane diffusion may compete with surface-to-bulk release, we will be able to estimate G in a model-independent manner. We will reveal the relative location of the proton pathway with respect to membrane structure by performing the first systematic investigation of proton`s residence time on the interface as a function of membrane surface and dipole potentials. These experiments will also allow dissection of the contributions of these two components to G. The accompanying analysis of electrostatics will help clarify whether the hydroxyl ion might substitute the proton as the migrating species. Testing the affinity of the amphiphilic hydronium to solutes with different hydrophobicities may explain why titratable groups are not required for stabilizing the proton at the surface. Finally, we will demonstrate the importance of the waters of lipid hydration for lateral proton migration by modifying the hydration shell e.g. with osmolytes. Establishing the molecular picture of interfacial proton migration will help to understand a large variety of proton-coupled transport processes.

Energy homeostasis of living organisms is intricately linked to proton transport. During photosynthesis in plants protons are pumped across the thylakoid membranes of chloroplasts. Higher organisms store energy by first using proton pumps to establish a proton concentration gradient across the inner mitochondrial membrane and second having ATP synthases exploiting this gradient to produce ATP, the energetic currency of the cell. The process is only efficient, when proton pumps and ATP synthases are tightly coupled. That is, efficiency requires protons to travel along the membrane instead of taking a detour across the inner space of the mitochondria. In the frame of this project we were asking the question what keeps the proton close to the membrane, i.e. what prevents proton surface-to-bulk release. We were able to show that proton binding by surface anchored moieties was not involved. On the contrary, the peculiar structure of surface water ensured that proton migration along membranes was very much favored as compared to the diffusion in the perpendicular direction. Theoretical models that envisioned the migrating proton to undergo equilibrium reactions with titratable surface groups did not explain our experiments. This observation is in marked contrast to the concept of proton antennae. The temperature dependence of proton diffusion between photo-releases spot and distant observation sites was only compatible with non-equilibrium kinetics. We also undertook a large effort to visualize how the water structure biases proton diffusion by performing ab initio molecular dynamics simulations. In addition, we identified small molecules that are able to alter interfacial water structure thereby inhibiting fast proton surface migration. Dismantling the concept of proton antennae enables a bias free investigation of the coupling mechanisms between membrane surface and proton channels or transporters.