Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
The mechanism of anion transport through the phosphate-starvation inducible OprP-channel of Pseudomonas aeruginosa outer membrane was studied in planar lipid bilayer membranes. The single-channel conductance of OprP was 160 pS in 100 mM chloride solution. Addition of other anions, in particular of phosphate, di and tribasic anions lead to a strong decrease of the chloride conductance. The decrease was used to calculate the stability constants for the binding of these ions to the binding site of the channel on the basis of a one-site two-barrier model. The stability constant of the binding of phosphate to the site was 11000 M-1 at neutral pH. Surprisingly, di-and tribasic anions, such as sulfate and citrate had a much lower affinity to the binding site inside the channel. Although the single-channel conductance was dependent on the external pH, the stability constants for phosphate binding decrease only slightly for increasing the pH. The use of negatively-charged lipids instead of neutral ones in the planar lipid bilayers had no influence on the single-channel conductance of the OprP-channel, suggesting that the channel is shielded from the influence of surrounding molecules. Its permeability properties are probably not influenced by negatively-charged lipopolysaccharide molecules.
The Journal of General Physiology, 1987
Protein P trimers isolated and purified from Pseudomonas aeruginosa outer membrane were reconstituted in planar lipid bilayer membranes from diphytanoyl phosphatidylcholine. The protein trimers formed highly anion-specific channels with an average single channel conductance of 160 pS in 0 .1 M Cl solution. A variety of different nonvalent anions were found to be permeable through the channel, which suggests a channel diameter between 0 .5 and 0.7 nm. The selectivity for the halides followed the Eisenman sequence AVI (without At-). The ion transport through the protein P channel could be explained reasonably well by a one-site, two-barrier model. The stability constant of the binding of CI-to the site was 20 M-' at neutral pH. The binding of anions to the site was pH dependent, which suggested that several charges are involved in the closely spaced selectivity filter. Permeability ratios for different anions as calculated from bi-ionic potentials showed agreement with corresponding ratios of single channel conductances. The protein P channels were not voltage-gated and had lifetimes of the order of several minutes. The current-voltage curves were linear for membrane potentials up to 150 mV, which suggested that Nernst-Planck-type barriers rather than Eyring barriers were involved in the movement of anions through the protein P channel .
Proceedings of the National Academy of Sciences, 1996
The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction ofprotons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (-15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction ofthe proton with this cluster is at a very fast rate (3.1010 M-t.s-t). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates ofthe cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.