The Dictyostelium genome contains a single rapA gene,which encodes a Rap1 monomeric G protein. As attempts at generating rapA-null Dictyostelium cells had been unsuccessful,expression of antisense RNA from the rapA gene under control of the folate repressible discoidin promoter was used to reduce cellular levels of the Rap1 protein. As Rap1 levels gradually decreased following antisense rapA RNA induction, growth rate and cell viability also decreased, a result consistent with the idea that rapA is an essential gene. The Rap1-depleted cells exhibited reduced viability in response to osmotic shock. The accumulation of cGMP in response to 0.4 M sorbitol was reduced after rapA antisense RNA induction and was enhanced in cells expressing the constitutively activated Rap1(G12V) protein, suggesting a role for Rap1 in the generation of cGMP. Dictyostelium Rap1 formed a complex with the Ras-binding domain of RalGDS only when it was in a GTP-bound state. This assay was used to demonstrate that activation of Rap1 in response to 0.4 M sorbitol occurred with initial kinetics similar to those observed for the accumulation of cGMP. Furthermore, the addition of 2 mM EDTA to osmotically shocked cells, a treatment that enhances cGMP accumulation, also enhanced Rap1 activation. These results suggest a direct role for Rap1 in the activation of guanylyl cyclase during the response to hyperosmotic conditions. Rap1 was also activated in response to low temperature but not in response to low osmolarity or high temperature.

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spinlabeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.

Folylpolyglutamate synthetase (FPGS) catalyzes the addition of glutamate to folate derivatives to form folate polyglutamates. FPGS is essential for folate biosynthesis in bacteria and retention of folate pools in eukaryotes. X-ray crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS suggest that binding of folate triggers a conformational change that activates FPGS. We used EPR and CD spectroscopy to further characterize the conformational change in the FPGS reaction. For EPR spectroscopy, two cysteine residues were introduced into FPGS by site-directed mutagenesis, K172C in the N-terminal domain and D345C in the C-terminal domain. The mutant protein was expressed, purified, and labeled with methanethiosulfonate. Addition of ATP, tetrahydrofolate, or 5,10-methylenetetrahydrofolate but not glutamate to FPGS showed broadening of EPR spectra, which is due to stronger spin-spin interactions, suggesting that both ATP and tetrahydrofolates cause a conformational change. ATP binding had an EPR spectrum distinct from that of tetrahydrofolate binding, indicating that it caused a different conformational change. When both ATP and THF were bound, the spectrum was identical to that seen when THF alone bound to the enzyme, showing that the THF-induced conformation was dominant. The spectral broadening suggests that the conformation change involves the two domains moving closer together, which is consistent with the rigid-body rotation of the C-terminal domain observed in the FPGS crystal structure with AMPPCP and 5,10-methylenetetrahydrofolate bound. No changes in the CD spectra were observed with the addition of FPGS substrates, suggesting that the conformational changes did not affect the secondary structure elements of the enzyme. These studies confirm the conformational change seen in the crystal structure by an independent method but also show that ATP binds to the free enzyme and affects its conformation.