Energy Sources for HCO3− and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 6251

Light-dependent inorganic C (Ci) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO2 or HCO3− uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular Ci pool of 20 mm, even though CO2 fixation was completely inhibited, indicating that cyclic electron flow was involved in the Ci-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar Ci accumulation was observed, suggesting that linear electron flow could support the transport of Ci. When carbonic anhydrase was not present, initial CO2 uptake was greatly reduced and the extracellular [CO2] eventually increased to a level higher than equilibrium, strongly suggesting that CO2 transport was inhibited and that Ci accumulation was the result of active HCO3− transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, Ci transport and accumulation were inhibited by inhibitors of CO2 transport, such as COS and Na2S, whereas Li+, an HCO3−-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, Ci transport and accumulation were not inhibited by COS and Na2S but were inhibited by Li+. These results suggest that CO2 transport is supported by cyclic electron transport and that HCO3− transport is supported by linear electron transport.

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria1

The induction of a high-affinity state of the CO2-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO2 concentrations were resuspended in low-CO2 buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO2-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO2 and HCO3− fluxes indicated that only the affinity of HCO3− transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO2 uptake remained unchanged. Fast induction of high-affinity HCO3− transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO3− transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO3− transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.

Scorpion toxins as natural scaffolds for protein engineering.

A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded beta-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and 1H NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10(-8) M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional means for design and engineering of small proteins.

A paradigm for drug discovery employing encoded combinatorial libraries.

Very large combinatorial libraries of small molecules on solid supports can now be synthesized and each library element can be identified after synthesis by using chemical tags. These tag-encoded libraries are potentially useful in drug discovery, and, to test this utility directly, we have targeted carbonic anhydrase (carbonate dehydratase; carbonate hydro-lyase, EC 4.2.1.1) as a model. Two libraries consisting of a total of 7870 members were synthesized, and structure-activity relationships based on the structures predicted by the tags were derived. Subsequently, an active representative of each library was resynthesized (2-[N-(4-sulfamoylbenzoyl)-4'-aminocyclohexanespiro]-4-oxo-7 -hydroxy- 2,3-dihydrobenzopyran and [N-(4-sulfamoylbenzoyl)-L-leucyl]piperidine-3-carboxylic acid) and these compounds were shown to have nanomolar dissociation constants (15 and 4 nM, respectively). In addition, a focused sublibrary of 217 sulfamoylbenzamides was synthesized and revealed a clear, testable structure-activity relationship describing isozyme-selective carbonic anhydrase inhibitors.