Title of Abstract
Identification of the Phosphorylation Sites on RitR
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
Iron is essential to the survival of nearly all known organisms. Free iron in the cell also acts as a catalyst, reacting with common oxygen species within the cell, creating hydrogen peroxide and hydroxyl radicals. Every organism has to have a way to control iron uptake, to inhibit excess levels of iron within the cell. In S. pneumonia, the iron uptake mechanism is activated by extracellular iron; however, the sensory mechanism used to inhibit this uptake is not yet well understood. When iron is sensed extracellularly, a complex known as Stk-P is activated, and in the presence of ATP, this molecule will phosphorylate RitR. When not phosphorylated, RitR is bound tightly to the DNA of S. pneumonia in close proximity to the piu (pneumococcal iron uptake operon), preventing transcription of that portion of the DNA. When phosphorylated, RitR is not bound to the DNA, allowing transcription to occur. This research focuses on the location of phosphorylation on RitR. This will help understand how this protein functions and how it interacts with the DNA. Additionally, this work explores the difference in binding between RitR in its purified form, and the modified version of RitR. The phosphorylation sites on RitR were identified, and the structure changes caused by this phosphorylation were also explored. The sites that are modified by the kinase were identified to be Ser-19, Tyr-163, Thr-168, and Ser-172. Based on a homology model, three of these sites are located on a single helix in the DNA binding domain, while the Ser-19 site is located on the opposite side of the protein. The effects of these sites and their modification on the function of the protein will be explored further by modifying these amino acids to prevent the kinase from phosphorylating at these positions. The effects will then be observed on DNA binding to determine how this would influence the protein as it interacts with the piu.
Honors Thesis Committee
Nicholas Grossoehme, Ph.D.; Jason Hurlbert, Ph.D.; and Fatima Amir, Ph.D.
Previously Presented/Performed?
Southeast Regional Meeting of the American Chemical Society (SERMACS), Charlotte, North Carolina, November 2017; South Carolina INBRE Symposium, Columbia, South Carolina, October 2017; Summer Undergraduate Research Experience (SURE) Symposia, Winthrop University, July and September 2017
Grant Support?
Supported by an REU grant from the South Carolina EPSCoR/IDeA Program, and by an SC INBRE grant from the National Institute of General Medical Sciences (NIH-NIGMS)
Start Date
20-4-2018 2:45 PM
Identification of the Phosphorylation Sites on RitR
West 219
Iron is essential to the survival of nearly all known organisms. Free iron in the cell also acts as a catalyst, reacting with common oxygen species within the cell, creating hydrogen peroxide and hydroxyl radicals. Every organism has to have a way to control iron uptake, to inhibit excess levels of iron within the cell. In S. pneumonia, the iron uptake mechanism is activated by extracellular iron; however, the sensory mechanism used to inhibit this uptake is not yet well understood. When iron is sensed extracellularly, a complex known as Stk-P is activated, and in the presence of ATP, this molecule will phosphorylate RitR. When not phosphorylated, RitR is bound tightly to the DNA of S. pneumonia in close proximity to the piu (pneumococcal iron uptake operon), preventing transcription of that portion of the DNA. When phosphorylated, RitR is not bound to the DNA, allowing transcription to occur. This research focuses on the location of phosphorylation on RitR. This will help understand how this protein functions and how it interacts with the DNA. Additionally, this work explores the difference in binding between RitR in its purified form, and the modified version of RitR. The phosphorylation sites on RitR were identified, and the structure changes caused by this phosphorylation were also explored. The sites that are modified by the kinase were identified to be Ser-19, Tyr-163, Thr-168, and Ser-172. Based on a homology model, three of these sites are located on a single helix in the DNA binding domain, while the Ser-19 site is located on the opposite side of the protein. The effects of these sites and their modification on the function of the protein will be explored further by modifying these amino acids to prevent the kinase from phosphorylating at these positions. The effects will then be observed on DNA binding to determine how this would influence the protein as it interacts with the piu.
