Дисертації: "Urea"

1

Gray, Lawrence Robert. "Detailed characterization of the urea channel urei from helicobacter pylori." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/3306.

Повний текст джерела

Анотація:

HpUreI is a pH-gated urea channel found in the pathogenic bacterium Helicobacter pylori. This protein is an essential component of the mechanism of acid acclimation, which allows Helicobacter pylori to survive in the acidic conditions of the stomach. HpUreI conducts urea into the cytoplasm, where it is hydrolyzed by urease into carbon dioxide and ammonia. These products then transit back into the periplasm, where they function as a buffer and proton consumer respectively. HpUreI is an attractive target for small molecule inhibitors for the treatment of H. pylori infections as mutant strains lacking this protein no longer survive under acidic conditions. Despite the importance of HpUreI, it remains biochemically uncharacterized and many questions remain as to how this channel performs its roles. We have solved many of the technical issues regarding the heterologous expression and purification of HpUreI, allowing us to investigate this protein in detail. A robust stopped-flow light-scattering assay was developed which was used to determine the permeability of urea (or other solute) through HpUreI reconstituted proteoliposomes. With slight modifications this assay was be used to measure a wide range of characteristics and variables. Our results show that HpUreI is a hexameric protein that has a relatively weak affinity for urea (˜160mM). Proteoliposome studies indicate that HpUreI is highly selective for urea and hydroxyurea, and is able to conduct water. Interestingly, water and urea conduction is pH-gated, suggesting that both solutes share a common conduction pathway. HpUreI displayed a pH-dependent activity profile with a pH of half maximum activity of ˜5.9. Based on these results an updated mechanism of acid acclimation was proposed. HpUreI is a pH-gated channel; only conducting urea under acidic conditions. The mechanism by which this occurs is not well understood, but can be localized to the periplasmic loops. Chimeric proteins were prepared by swapping the periplasmic loops of HpUreI and StUreI, a pH-independent UreI channel from Streptococcus thermophilus. Our results show that the pH-gating behavior of HpUreI was lost if either periplasmic loop was replaced with the corresponding loop from StUreI. Conversely, pH-gating was gained by StUreI when both periplasmic loops were swapped for those of HpUreI. A model of pH-gating was proposed which takes these findings into account. The mechanism of urea conduction was also examined. The recent crystal structure of HpUreI revealed a ladder of tryptophan residues lining one face of the conduction pathway. Mutation of these residues resulted in lower rates of urea conduction and reduced urea affinity. Our findings indicate that urea interacts directly with the tryptophan residues, via stacking and dipole-dipole interactions, to facilitate urea conduction. These studies have greatly increased our understanding of HpUreI and the role it plays in H. pylori. Further research is required to fully elucidate the mechanisms by which HpUreI operates. However, this is a starting point with which to pursue the ultimate goal of developing small molecule drugs to inhibit HpUreI, culminating in the eradication of H. pylori infections and prevention of gastric cancer.