Halorhodopsin is a light-driven chloride anion pump in which the trans-->cis photoisomerization of a retinal chromophore triggers a photocycle resulting in the translocation of chloride across the plasma membrane. The mechanism of chloride transfer past the cis retinal is determined here by computing multiple pathways for this process. The calculations reveal two conditions of the valve mechanism. First, a lumen absent in the ground state structure is transiently opened by chloride passage. Second, this activated opening, which is achieved by flexible deformation of the surrounding protein, is shown to significantly raise the chloride translocation barrier between photocycles, thus preventing chloride backflow. Unlike macroscopic valve designs, the protein allows differential ion flows in the pumping and resting states that are tuned to match the physiological timescales of the cell, thus creating a "kinetic" valve.

Surface salt bridges are ubiquitous in globular proteins. Their contribution to protein stability has been extensively debated in the past decade. Here, molecular dynamics simulations are performed starting from a non-equilibrium state of Staphylococcal nuclease (SNase) with C-terminal truncation (SNaseDelta). The results indicate a key role in the unfolding of the surface salt bridge between arginine 105 and glutamate 135. Experimentally, SNaseDelta is known to be partially unfolded. However, in simulations over 1 ns at 300 K and over 500 ps at 400 K, SNaseDelta remains stable in the native-like folded conformation, the salt bridge hindering unfolding. When the potential function is altered so as to selectively weaken the salt bridge, which then breaks rapidly at 430 K, the protein starts to unfold. The results suggest that breaking of this salt bridge presents a significant barrier to the unfolding transition of SNaseDelta from a native-like state to the unfolded state. Potential of mean force calculations indicate that the barrier height for this transition is approximately 7 kcal/mol.

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The light-driven pump Halorhodopsin (hR) uses the energy stored in an initial meta-stable state (K), in which the bound retinal chromophore has been photoisomerized from all-trans to 13-cis, to drive the translocation of one chloride anion across the membrane. Thus far it was unclear whether retinal twisting or charge separation between the positive Schiff base of the retinal and the chloride anion is the primary mechanism of energy storage. Here, combined quantum mechanical/molecular mechanical (QM/MM) simulations show that the energy is predominantly stored by charge separation. However, a large variability in retinal twisting is observed, thus reconciling the contradictory hypotheses for storage. Surprisingly, the energy stored in the K-state amounts to only one-fifth of the photon energy. We explain why the protein cannot store more: even though this would accelerate chloride pumping, raising the K-state also reduces the relative energy barriers against unproductive decay, in particular via the premature cis to trans backisomerization. Indeed, the protein has maximized its storage so that the back-isomerization barriers are just high enough (>18 kcal/mol) to keep the decay rate (1/100 ms) slower than the remaining photocycle (1/20 ms). This need to stabilize the captured photon-energy until it can be used in subsequent steps is inherent to light-driven proteins.Light driven pumps such as halorhodopsin (hR) 2 stand apart from other energy-driven transporters. To them, the primary source of energy is not permanently available, unlike energy stored in the form of chemical bonds (for example, ATP in the case of the Na ϩ /K ϩ -ATPase (1)) or in electrochemical transmembrane gradients (like the Na ϩ /glucose symport transporter (2)). Thus, they must be able to first harvest the energy of the photon, before converting that energy into work. The harvesting of the fleeting photon necessitates converting the photon energy into a meta-stable form of molecular energy, and to prevent the dissipation of this molecular energy until it can be subsequently used to power the pumping mechanism. In other words, the protein must be able to simultaneously store and stabilize the energy. For the absorption of light, proteins employ a chromophoric ligand or group, which changes from one state to another upon photon absorption. This transition is unfavorable and slow in the electronic ground-state, but is favored when the chromophore has been excited by the photon. After the return of the system to the electronic ground-state (within picoseconds in the case of hR (3)), the chromophore relaxes either to the starting ground-state conformation (thus reducing the quantum yield) or to a meta-stable high-energy state (with a quantum yield of 34% in the case of hR (3)). The energy stored in this meta-stable form relative to the groundstate form is what powers the subsequent pumping steps (Fig. 1A). Understanding how this energy is stored thus constitutes an essential aspect of the mechanism and has been the focus of studies on visual rh...

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