Quantitation of physical-chemical properties of the aqueous phase inside the phoE ionic channel.

The anion-specific channel of the phoE porine is a miniature body of water surrounded by peptide walls. The physical and chemical properties of the water in such a microscopic space were measured by monitoring the dynamics of a well-studied reaction--the protolytic dissociation of a strong acid. To attain this purpose, we allowed pyranine (8-hydroxypyrene-1,3,6-trisulfonate) to bind to the anion-specific channel. The dye is bound, with a 1:1 stoichiometry, with a delta G = -9.5 kcal/mol. Photoexcitation of the dye, to its first electronic singlet state (phi OH*), renders it very acidic and the hydroxyl proton dissociates to H+ and excited anion (phi O*-). We employed single photon-counting time-resolved fluorimetry, to monitor the reversible dissociation of pyranine as it proceeds within the channel and reconstructed the observed signal by a numerical integration of the differential diffusion equation pertinent for a proton within the channel. The most characteristic feature of the water-filled channel, is the intensified electrostatic interactions attained by the low dielectric constant of the diffusion space, epsilon eff = 24. For this reason, the electric field of a few positive charges is sufficient to ensure that an anion entering the channel will be effectively sucked in. The interaction of the water molecules with the peptide structure forming the channel affects the physical properties of the water. Their capacity to conduct proton, quantitated by the protons diffusion coefficient (4.5.10(-5) cm2/s), is reduced by 50% with respect to that of bulk water. The activity of the water in the channel is reduced to alpha H2O = 0.966. These observation are in accord with our previous studies of water in small defined cavities in proteins.