Thus, electrostatic repulsions between Ganetespib N- and C-terminal domains force the protein into the “”open”" position. This in turn releases the N-terminal domain,
forming a stable complex with KdpE~P and the DNA [25] initiating kdpFABC expression. Replacement of the KdpD-Usp domain with UspF or UspG results in inversion of the surface net charges. The negative net surface charge of these two proteins forces electrostatic attraction between the N- and the C-terminal regions, leaving KdpD in the “”OFF”" state under all conditions. Conclusion The Usp domain within KdpD is important for proper KdpD/KdpE signaling. Alterations within this domain can completely prevent the response towards K+ limitation as well as salt stress. The KdpD-Usp domain surface contains numerous positively charged amino acids. Electrostatic repulsion and attraction between the N-terminal and C-terminal domain are supposed to be important for KdpD (de)activation. Therefore, selleck the KdpD-Usp domain not only functions as a binding surface for the native scaffold UspC, but also seems to be crucial
for internal KdpD signaling, shifting the protein from an “”OFF”" into an “”ON”" state. Methods Materials [γ32-P]ATP and NAP-5 gel filtration columns were purchased from Amersham GE Healthcare. Goat anti-(rabbit IgG)-alkaline phosphatase was purchased from Biomol. All other reagents were reagent grade and obtained from commercial sources. Baf-A1 cell line Bacterial strains and plasmids E. coli strain JM 109 [recA1 endA1 gyrA96 thi hsdR17 supE44λrelA1 Δ(lac-proAB)/F'traD36 proA + B + lacI q lacZΔM15] Progesterone [30] was used as carrier for the plasmids described. E. coli strain TKR2000 [ΔkdpFABCDE trkA405 trkD1 atp706] [31] containing different
variants of plasmid pPV5-3 encoding the different KdpD-Usp derivatives (see below) was used for expression of the kdp-usp derivatives from the tac promoter. E. coli strain HAK006 [ΔkdpABCD Δ(lac-pro) ara thi] [32] carrying a kdpFABC promoter/operator-lacZ fusion was used to probe signal transduction in vivo. E. coli LMG194 [F- ΔlacX74 galE galK thi rpsL ΔphoA (PvuII) Δara714leu::Tn10] [33] was used for expression of the kdp-usp derivatives from the araBAD promoter. To replace the Usp domain in E. coli KdpD with the E. coli Usp protein sequences, the corresponding usp genes were PCR amplified using genomic DNA of E. coli MG1655 [34] as a template. The uspA, uspD, uspE, uspF, and uspG genes were amplified with primers complementary at least 21 bp to the 5′ or the 3′ ends of the corresponding genes with overhangs for a 5′ NsiI site and a 3′ SpeI site, respectively. uspC was amplified similarly, but with a 5′ terminal SacI site.
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