Abstract
We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. N(alpha)-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 +/- 20 microg/ml. The compounds were shown to compete with fluorophore-labeled sigma(32)-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.
Highlights
Together with its co-chaperones the de novo protein folding of ϳ20% of the Escherichia coli proteins larger than 30 kDa (1– 4)
Inhibitor-related Viability after Severe Heat Shock—To judge whether different cellular levels of DnaK influence the antibacterial activity of compound 1, we have introduced the plasmid pTrc99A-DnaK expressing DnaK under the control of an IPTG-inducible promoter into E. coli MC4100 ⌬dnaK cells (Fig. 5)
In this study we have used inhibition analyses to characterize a molecular pathway by which DnaK triggers protein folding in E. coli cells
Summary
Materials—Monoclonal anti-DnaK antibody was purchased from Stressgen (Victoria, Canada). The erythrocytes (2.5 ϫ 108 cells/ml) were treated with fatty acylated benzamido derivatives at final concentrations ranging from 3.1 to 50 g/ml and incubated under gently shaking at 42 °C for 30 min. The washed pellets were again resuspended in 320 l of buffer B by sonification, subsequently 80 l of 10% (v/v) Nonidet P-40 was added, and aggregated proteins were isolated by centrifugation (15,000 ϫ g, 20 min, 4 °C). Controls showed 0.1 l of E. coli lysate and 0.2 g if recombinant DnaK
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
