Electron paramagnetic resonance (EPR) spectroscopy is a powerful and versatile spectroscopic tool for detecting unpaired electrons in molecular systems. We are particularly interested in using EPR to study protein structure and dynamics. By attaching two spin-labels, which contain unpaired electrons, to different regions of a protein, we can measure the distance between the spin-labels to gain insight of protein conformations. The signal sensitivity is limited by protons on amino acids adjacent to the spin-label. Coupling of the spin-label to these nearby protons causes decay of the EPR signal intensity. This decay is quantified by the phase memory time (T_M). While it is known that the presence of neighboring protons results in a shorter T_M, the quantitative effect these protons have on T_M is not well understood. To understand this effect, I utilize maltose-binding protein (MBP) as a model protein. I place spin-labels on various regions of MBP via mutations and site-directed spin labeling to determine how specific neighboring amino acids affect T_M. Spin-labels at MBP sites with a low number of surrounding protons are expected to have a longer T_M than spin-labels at sites with more surrounding protons. Measuring T_M values of different MBP mutants helps further our understanding of how local protein environments impact signal intensity, enabling us to develop quantitative models to predict T_M values in silico. Such models can be used to computationally determine ideal spin-label sites for proteins that previously could not be studied using EPR due to lack of signal sensitivity, in particular membrane proteins.

The modification of tRNA plays a significant role in the efficiency and accuracy of translation during protein synthesis. A modification that plays a direct role in reading cognate codons of mRNA in E. coli is the 5-oxyacetic acid methyl ester (mcmo5) modification. This modification occurs on the uracil base at position 34 (U34). The biosynthetic pathway of this modification is initiated via a hydroxylation reaction. Previous in vivo studies demonstrate the enzyme TrhP, tRNA hydroxylation protein, performs this hydroxylation reaction in anaerobic conditions. No in vitro work has been done to study this enzyme and its mechanistic function. TrhP is known to coordinate an iron-sulfur cluster, a metallic cofactor known to contribute to a variety of critical cellular processes, however, the necessity of an iron-sulfur cluster for a hydroxylation reaction is unique to this newly discovered protein family. The goal of this research project is to spectroscopically characterize TrhP’s iron-sulfur cluster to understand the importance of the FeS cluster. Site-directed mutagenesis is utilized to study the coordination of the iron-sulfur cluster. Changes to iron-sulfur cluster coordination are monitored via UVVIS, electron paramagnetic resonance (EPR), and colorimetric assays. These experiments determine how the loss of cysteine, a known iron-sulfur cluster ligand, impacts the iron-sulfur cluster coordination. Coordination of a [2Fe2S] cluster by 4 conserved cysteines is expected, and UVVIS data agrees with that hypothesis. Colorimetric assays show the cysteine to alanine mutants contain less iron than wild-type TrhP, indicating each cysteine has a significant role in cluster binding. Learning more about the specific coordination will establish the site of cluster-binding within TrhP and shed light on the cluster’s role in TrhP’s stability, geometry, and redox properties which all contribute to the enzyme’s modification activity.