In addition to a genetic macromolecule and a semipermeable membrane, a source of energy to drive intracellular processes must be present. A common source of free energy used in many cells to drive unfavorable processes is a proton gradient, whose formation in modern cells can be coupled to energy input from oxidation, ATP cleavage, light, or the collapse of another gradient. Could a proton gradient be formed in protocells? It can, quite easily, when coupled to the growth of fatty acid vesicles. If a fatty acid vesicle is to grow, more fatty acid must be added to the outer leaflet. The protonated, uncharged form of the fatty acid would preferentially be added, since it would lead to less electrostatic repulsion between adjacent head groups. The protonated, uncharged form of the fatty acid would also be most likely to flip to the inner leaflet to minimize stress asymmetries in the leaflets. Once in the inner leaflet, it could deprotonate to form H+(aq) in the inside of the membrane, creating a transmembrane proton gradient and transmembrane potential. The energy released on growth of the membrane is partly captured in the formation of a proton gradient, as shown in the figure below.
The proton gradient would soon inhibit its own formation since further movement of protons into the cell would be attenuated by the positive transmembrane potential unless metal ions inside moved outside. In addition, the gradient would collapse after growth stopped. The investigators made fatty acids vesicles in the presence of pH 8.5 buffers whose pH was adjusted with an alkali metal hydroxide. The external pH was reduced to 8.0, resulting in a 0.5 pH unit proton concentration gradient. (Changes in intravesicular pH were measured with pH-sensitive fluorophore, HPTS.) Inward movement of protons down a concentration gradient, as shown in the figure below, would occur with time, collapsing the imposed concentration gradient.
With fatty acid vesicles, this artificial pH gradient collapsed quickly, suggesting the vesicle permeability to protons was high. The rate was too high for simple flip-flop diffusion. Inward movement of protons appeared to be facilitated by outward movement of the M+ ions. The rate of decay of the proton gradient was exponential, and the resulting first order rate constant was easily determined. A graph of the rate constant for pH gradient collapse vs unsolvated ionic radius of M+ decreased with increasing radius (i.e. kNa > kK > KRb > KCs, suggesting that the pH gradient would be more stable if large, impermeable or otherwise trapped cations were encapsulated. When vesicles were made with encapsulated Arg+, the imposed pH gradient did not collapse for hours. If oleic acid micelles were added to oleic acid vesicles with encapsulated Arg+, with no artificial pH gradient induced across the membrane, the vesicle grew with concomitant movement of protons into the vesicle, producing a pH gradient of 0.3 within seconds.
These experiments show that membrane growth and energy storage could be coupled, and the right composition of encapsulated material could lead to a stable transmembrane pH gradient, a source of energy to drive biological processes. It even suggests that a charge polyanion would be beneficial as a genetic carrier.
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