Professor Menachem Gutman
Laser Laboratory for Fast Reactions in Biology
Tel Aviv University
Israel

Friday, October 15, 1999
3:00 pm (CT)
3269 Beckman Institute

Abstract

The passage of ions through a single file of water molecules, like the gramicidin channel, has only recently advanced up to the state of modeling the passage of protons. The flux in large-pore channels, likes the porins or the VDAC, is even more complex. The electrostatic potential within the channel varies not only along the channel but also across it. As a result the channels exhibit ion selectivity and the measured flux is less than predicted by the conductivity of the electrolyte solution. In the present study we have investigated the dynamics of ion passage through the large-pore channel, PhoE , using a combination of electrostatic potential mapping and chemical dynamics calculations. The three dimensional structure of large-pore channels, derived by crystallographic studies, is not sufficient for the prediction of ion fluxes. Both the dynamics of the protein structure and the quantitation of the dielectric constant of the intra-cavity water phase are pre-requisite for reconstruction of the ion flux. Molecular Dynamics of porins indicated that the structural fluctuations in the eyelet region are much faster than the passage time of ions, thus the average state of the channel can be used as a fair approximation for predicting the conductance of the channel. As for the dielectric constant, theoretical calculations indicated that the intra-cavity of beta-barrel structures will have epsilon‰40. In our studies we examined time-resolved fluorescence of pyranine anchored to the PhoE protein. The dye was excited by a ps laser pulse and the fluorescence dynamics revealed the reversible dissociation of the proton from the excited molecule. The measured transients were analyzed as a geminate recombination reaction proceeding in a non-homogeneous electric field. We combined the electrostatic potential, calculated by Delphi, with the geminate recombination simulations of Agmon to determine the dielectric constant of the intra-cavity and to simulate the electro-dynamics properties of the large-pore channel. A major advantage of this mode of analysis is that it accounts for the electrostatic potential along the whole length of the channel rather than only in the eyelet zone. The trajectory of the proton escape out of the channel was derived and the electrostatic gradients were fed into the proton propagation program. Is was found that the measured dynamics could be reconstructed with a dielectric constant of the intra-cavity space of ‰25. On the basis of these parameters the flux of negative and positive ions along the channel were reconstructed, reproducing both the rate and the ionic-selectivity of the channel.

Tea and coffee will be served in R3151 Beckman Institute at 2:15pm.