A Unifying Concept for Ion Translocation by Retinal Proteins

First, halorhodopsin is capable of pumping protons after illumination with greenand blue light in the same direction as chloride. Second, mutated bacteriorhodopsin where the proton acceptor Asp85 and the proton donor Asp96 are replaced by Asn showed proton pump activity after illumination with blue light in the same direction as wildtype after green light illumination. These results can be explained by and are discussed in light of our new hypothesis: structural changes in either molecule lead to a change in ion affinity and accessibility for determining the vectoriality of the transport through the two proteins

Light-driven proton or chloride pumping by halorhodopsin

Halorhodopsin from Halobacterium halobium was purified and reconstituted with lipids from purple membranes. The resulting protein-containing membrane sheets were adsorbed to a planar lipid membrane and photoelectric properties were analyzed. Depending on light conditions, halorhodopsin acted either as a light-driven chloride pump or as a proton pump: green light caused chloride transport and additional blue light induced proton pumping. In the living cell, both to these vectorial processes would be directed toward the cytoplasm and, compared to ion transport by bacteriorhodopsin, this is an inversed proton flow. Azide, a catalyst for reversible deprotonation of halorhodopsin, enhanced proton transport, and the deprotonated Schiff base in the 13-cis configuration (H410) was identified as the key intermediate of this alternative catalytic cycle in halorhodopsin. While chloride transport in halorhodopsin is mediated by a one-photon process, proton transport requires the absorption of two photons: one photon for formation of H410 and release of a proton, and one photon for photoisomerization of H410 and re-formation of H578 with concomitant uptake of a proton by the Schiff base

Roles of cytoplasmic arginine and threonine in chloride transport by the bacteriorhodopsin mutant D85T.

In the light-driven anion pump halorhodopsin (HR), the residues arginine 200 and threonine 203 are involved in anion release at the cytoplasmic side of the membrane. Because of large sequence homology and great structural similarities between HR and bacteriorhodopsin (BR), it has been suggested that anion translocation by HR and by the chloride-pumping BR mutant BR-D85T occurs by the same mechanism. Consequently, the functions of the R200/T203 pair in HR should be the same as those of the corresponding pair in BR-D85T (R175/T178). We have put this hypothesis to a test by creating two mutants of BR-D85T in which R175 and T178 were replaced by glutamine and valine, respectively. Chloride transport activities were essentially the same for all three mutants, whereas chloride binding and the kinetics of parts of the photocycle were markedly affected by the replacement of T178. In contrast, the consequences of mutating R175 proved to be less significant. These findings are consistent with evidence obtained on HR and therefore support the idea that the respective mechanistic roles of the cytoplasmic arginine/threonine pairs in HR and BR-D85T are equal

Bacteriorhodopsin mutants D85N, D85T and D85,96N as proton pumps

Proton translocation in the BR mutants D85N, D85T and D85,96N was studied by attachment of purple membranes to planar lipid bilayers. Pump currents in these mutants were measured via capacitive coupling and by use of the appropriate ionophores. All mutants have a reduced pK of their Schiff bases around 8–8.5 in common. At physiological pH, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) coexists. Excitation with continuous blue light induces in all three mutants an outwardly directed stationary pump current. These currents are enhanced upon addition of azide in D85N and D85,96N by a factor of 50, but no azide enhancement is observed in D85T. Yellow light alone induces transient inwardly directed currents in the mutants but additional blue light leads to a stationary current with the same direction. All the observed currents are carried by protons, so that the consecutive absorption of a yellow and a blue photon leads to inverted stationary photocurrents by the mutants, as observed with halorhodopsin (HR). A mechanistic model describing the inversion of proton pumping is discussed by the cis-trans, trans-cis isomerization of the retinal and the different proton accessibility of the Schiff base from the extracellular or the cytoplasmic side of the membrane

Estimation of the Production Rates of Serum Aminoterminal Propeptide of Type III Procollagen and Laminin in Human Fibrotic Liver

Peer Reviewe

Electrical Currents of the light driven Pump Bacteriorhodopsin. The Role of Asp 85 and Asp 96 on Proton Translocation

The functional properties of the light-driven proton pump of bacteriorhodopsin and two point-mutated analogues have been studied on planar lipid membranes and on gels where membrane fragments containing the ion pumps have been oriented in an electric field. It is shown that Asp 85 and Asp 96 playa central role in proton pumping of bacteriorhodopsi