Characterizing the Effect of Conformational Changes in the Protein SufU on its Ability to Enhance Enzymatic Activity of the Cysteine Desulfurase SufS in Streptococcus mutans
Date of Award
Doctor of Philosophy (Ph.D.)
Andrew T. Torelli (Advisor)
Alexander Izzo (Other)
George S. Bullerjahn (Committee Member)
H. Peter Lu (Committee Member)
Iron-sulfur (Fe-S) clusters are ancient cofactors that participate in numerous cellular processes, such as electron transfer, enzyme catalysis, redox sensing, DNA repair and gene regulation. To achieve high degree of regulation and control of free sulfide and iron ion three biosynthetic machineries were identified to assemble Fe-S clusters: ISC, SUF, and NIF. Scaffold protein represents a centerpiece of the biosynthetic pathway with two important roles. It must support the assembly of Fe-S clusters from constituent iron and sulfide species donated by other proteins followed by transfer of the nascent Fe-S clusters to other partners requiring clusters for their function. These two roles present an interesting conundrum, namely that scaffold proteins must initially exhibit robust affinity for Fe-S clusters as required for their assembly, and yet transfer of the Fe-S clusters to recipient proteins indicates lower affinity. A model explaining this apparent paradox is based on the ability of IscU, a well-known scaffold protein, to adopt two different conformational states with different affinities for Fe-S clusters and biosynthesis protein binding partners. During sequential steps of the Fe-S cluster biosynthesis cycle, IscU alternates its conformational state and affinity for Fe-S clusters, in order to accomplish Fe-S cluster assembly and transfer. Our interest is focused on the U-type protein from pathogenic Gram-positive bacteria Streptococcus mutans that belongs to a truncated version of typical SUF system represented by sufCDSUB operon. Even though, the SufU protein shares high sequence similarity with above mentioned IscU, it exhibits important distinguishing feature of an extra 19 amino acids that is denoted as the Gram-Positive Region (GPR). This dissertation is focused on the effect of conformational change of the scaffold protein SufU on the kinetic activity of its binding partner cysteine desulfurase SufS from Streptococcus mutans. Our aim is to characterize cysteine desulfurase behavior and relevant activity in the presence or absence on the sulfide acceptor protein present in different conformations achieved by single point site-directed mutagenesis in highly conserved residues in the GPR. Introduced mutations were shown to alter SufU structural conformation and biological function as the enhancer of cysteine desulfurase activity indicating the GPR region importance in structural variability and presumably in the protein-protein interactions.
Bauman, Mariia A., "Characterizing the Effect of Conformational Changes in the Protein SufU on its Ability to Enhance Enzymatic Activity of the Cysteine Desulfurase SufS in Streptococcus mutans" (2016). Photochemical Sciences Ph.D. Dissertations. 86.