Structure and function of a bacterial redox sensor.
(2016) KEMT10 20151Department of Chemistry
- Abstract
- Gram-positive bacteria e.g. Streptomyces coelicolor [8], Bacillus subtilis [43] and Staphylococcus aureus [40] contain a novel sensor for the nicotinamide adenine dinucleotide (NAD) redox balance that functions as a transcriptional repressor called Rex (redox). Rex plays a key role in regulating genes in energy metabolism. It also modulates transcription of important genes for cellular redox homeostasis including genes for alternative metabolic pathways and oxidative stress responses [42]. The current Rex structures have provided novel insights into dinucleotide regulation of gene expression. However, to obtain a deep and complete understanding, additional Rex structural forms are required. In this project wild type and mutant variants of... (More)
- Gram-positive bacteria e.g. Streptomyces coelicolor [8], Bacillus subtilis [43] and Staphylococcus aureus [40] contain a novel sensor for the nicotinamide adenine dinucleotide (NAD) redox balance that functions as a transcriptional repressor called Rex (redox). Rex plays a key role in regulating genes in energy metabolism. It also modulates transcription of important genes for cellular redox homeostasis including genes for alternative metabolic pathways and oxidative stress responses [42]. The current Rex structures have provided novel insights into dinucleotide regulation of gene expression. However, to obtain a deep and complete understanding, additional Rex structural forms are required. In this project wild type and mutant variants of Rex from Streptococcus agalactiae were expressed in Escherichia coli. The wild type Rex (with or without a His6-tag) was purified, and the affinity to NADH was measured with a novel technique called MicroScale Thermophoresis (MST). To test the effect of mutation on the dissociation constant (Kd) of Rex for NADH binding, the following mutations were investigated; His6-Rex-triple mutant (S158A, E160A and S180T) and two single mutations; His6-Rex-R24S and His6-Rex-D130R. MST analysis revealed that the Kd value for Rex/NADH binding was ~5 µM while, the measured Kd for His6-Rex/NADH binding was ~50 µM. The difference in the measured Kd values might be due to an effect of the His6-tag. (Less)
- Popular Abstract
- Bacteria have a fantastic ability to adapt to environmental changes. They can synthesize a wide variety of proteins, which can control different situations. One situation is when bacteria encounter stress. Stress is a chemical or physical environmental change that can affect important cellular physiological processes or can cause macromolecular damage. Bacteria respond by cellular stress responses to increase the tolerance towards stress and adapt to it. Pathogenic bacteria are under constant stress when they are facing the host defense response. Understanding mechanisms of bacterial stress responses could help us in finding new antimicrobial targets and new ways to control infectious diseases. Gram-positive bacteria contain a novel sensor... (More)
- Bacteria have a fantastic ability to adapt to environmental changes. They can synthesize a wide variety of proteins, which can control different situations. One situation is when bacteria encounter stress. Stress is a chemical or physical environmental change that can affect important cellular physiological processes or can cause macromolecular damage. Bacteria respond by cellular stress responses to increase the tolerance towards stress and adapt to it. Pathogenic bacteria are under constant stress when they are facing the host defense response. Understanding mechanisms of bacterial stress responses could help us in finding new antimicrobial targets and new ways to control infectious diseases. Gram-positive bacteria contain a novel sensor for the nicotinamide adenine dinucleotide (NAD) reduction-oxidation balance in the cell, called Rex (redox). Rex plays a key role in regulating genes in energy metabolism and oxidative stress responses. To fully understand how this protein works we need detailed information on structure and function relationships of Rex. In this study, Rex from the pathogenic bacterium Streptococcus agalactiae was produced in Escherichia coli. The wild type and modified variants of Rex was purified and the effect of specific mutations were studied. This work lay the ground for future studies on Rex to provide an understanding of Rex structure and function properties. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8893998
- author
- Ahmad, Marwaa LU
- supervisor
- organization
- course
- KEMT10 20151
- year
- 2016
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Rex protein, Microscale thermophoresis (MST), dissociation constant (Kd), protein science, proteinvetenskap
- language
- English
- id
- 8893998
- date added to LUP
- 2019-02-26 10:54:45
- date last changed
- 2019-02-26 10:54:45
@misc{8893998,
abstract = {{Gram-positive bacteria e.g. Streptomyces coelicolor [8], Bacillus subtilis [43] and Staphylococcus aureus [40] contain a novel sensor for the nicotinamide adenine dinucleotide (NAD) redox balance that functions as a transcriptional repressor called Rex (redox). Rex plays a key role in regulating genes in energy metabolism. It also modulates transcription of important genes for cellular redox homeostasis including genes for alternative metabolic pathways and oxidative stress responses [42]. The current Rex structures have provided novel insights into dinucleotide regulation of gene expression. However, to obtain a deep and complete understanding, additional Rex structural forms are required. In this project wild type and mutant variants of Rex from Streptococcus agalactiae were expressed in Escherichia coli. The wild type Rex (with or without a His6-tag) was purified, and the affinity to NADH was measured with a novel technique called MicroScale Thermophoresis (MST). To test the effect of mutation on the dissociation constant (Kd) of Rex for NADH binding, the following mutations were investigated; His6-Rex-triple mutant (S158A, E160A and S180T) and two single mutations; His6-Rex-R24S and His6-Rex-D130R. MST analysis revealed that the Kd value for Rex/NADH binding was ~5 µM while, the measured Kd for His6-Rex/NADH binding was ~50 µM. The difference in the measured Kd values might be due to an effect of the His6-tag.}},
author = {{Ahmad, Marwaa}},
language = {{eng}},
note = {{Student Paper}},
title = {{Structure and function of a bacterial redox sensor.}},
year = {{2016}},
}