Exploration of RitR Oxidation and Dimerization
Poster Number
46
Session Title
Poster Session 2
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
All organisms need to maintain redox homeostasis through establishment of a normal steady state and redox signaling by using a broad range of mechanisms and redox sensing transcription factors. Surprisingly, none of the common redox-sensing transcription factors have been identified in streptococci. However, RitR (Repressor of iron transport Regulator), a protein originally identified due to its role in iron homeostasis in Streptococcus pneumonia, has emerged as the founding member of a novel family of redox sensors in prokaryotes. In the originally proposed model of RitR function, there is failure to demonstrate a mechanism for sensing intracellular iron concentrations, which is critical for metal and redox homeostasis. Recently, Glanville et al found a single cysteine (Cys128) redox switch in the linker domain and its oxidation state influences pneumococcal growth in an oxygen-dependent manner and that it represses the pneumococcal iron uptake (piu) operon in the oxidized form and reduces the amount of intracellular free ferrous iron. The crystallographically determined structure of RitR, in the oxidized and reduced state, suggests that the regulatory mechanism is dependent on the unraveling of helices and formation of an inter-protein disulfide bridge, allowing DNA binding and transcriptional repression of the piu system. In vivo , H2O 2 is the primary oxidant that drives this oxidation and dimerization. To better understand the role that the phosphorylation has on the function of the protein, this project aims to generate singly-phosphorylated RitR (spRitR) constructs with phosphoserine at each of the phosphorylation sites, using nonstandard amino acid (NSAA) technology. The degree to which site directed phosphorylation impacts oxidation will be assessed for each of the spRitR constructs and compared to the fully modified and unmodified forms of the protein.
Start Date
15-4-2022 12:00 PM
Exploration of RitR Oxidation and Dimerization
All organisms need to maintain redox homeostasis through establishment of a normal steady state and redox signaling by using a broad range of mechanisms and redox sensing transcription factors. Surprisingly, none of the common redox-sensing transcription factors have been identified in streptococci. However, RitR (Repressor of iron transport Regulator), a protein originally identified due to its role in iron homeostasis in Streptococcus pneumonia, has emerged as the founding member of a novel family of redox sensors in prokaryotes. In the originally proposed model of RitR function, there is failure to demonstrate a mechanism for sensing intracellular iron concentrations, which is critical for metal and redox homeostasis. Recently, Glanville et al found a single cysteine (Cys128) redox switch in the linker domain and its oxidation state influences pneumococcal growth in an oxygen-dependent manner and that it represses the pneumococcal iron uptake (piu) operon in the oxidized form and reduces the amount of intracellular free ferrous iron. The crystallographically determined structure of RitR, in the oxidized and reduced state, suggests that the regulatory mechanism is dependent on the unraveling of helices and formation of an inter-protein disulfide bridge, allowing DNA binding and transcriptional repression of the piu system. In vivo , H2O 2 is the primary oxidant that drives this oxidation and dimerization. To better understand the role that the phosphorylation has on the function of the protein, this project aims to generate singly-phosphorylated RitR (spRitR) constructs with phosphoserine at each of the phosphorylation sites, using nonstandard amino acid (NSAA) technology. The degree to which site directed phosphorylation impacts oxidation will be assessed for each of the spRitR constructs and compared to the fully modified and unmodified forms of the protein.
