Structure and biochemistry of iron-sulfur cluster biosynthesis proteins

Research in the Torelli lab comprises three core initiatives. Our first endeavor focuses on the interplay between molecular structure and function in proteins that biosynthesize iron-sulfur (Fe-S) clusters in bacteria. Fe-S clusters are essential protein cofactors required in all kingdoms of life owing to flexible properties that facilitate electron transport (e.g. within photosynthesis, respiration), environmental sensing, redox homeostasis, and even enzymatic catalysis as Lewis acids) 1. Our research builds on growing awareness that central proteins in the bacterial Fe-S biosynthesis pathways undergo structural variations during the biosynthesis cycle, for example between fully structured and partially disordered states 2. The equilibrium between these two states is influenced by metal binding and appears to modulate affinity for Fe-S clusters and other partner biosynthesis proteins 2,3. The effect of metal ion binding in particular raises interesting questions about possible mechanisms for the toxic effects of heavy metals that may disrupt the biosynthesis of essential Fe-S clusters with widespread downstream effects. We seek to measure the effects of non-cognate metals on the structures of Fe-S biosynthesis proteins as well as their impact on protein function. Recent results indicate homologous Fe-S biosynthesis proteins from representative Gram-negative and Gram-positive bacterial species discriminate between toxic transition metal ions, and, when binding to a non-cognate metal ion does occur, the resulting protein complex is altered in its ability to enhance the enzymatic activity of a binding partner.

Novel Blue Copper Protein Electron Carriers From Ammonia Oxidizing Archaea

Our second research initiative began recently and seeks to characterize novel electron-carrier proteins that support ammonia oxidation mediated by Archaea in the environment. Over the past 15 years, ammonia oxidizing Archaea (AOA) have been shown to exert dominant control over a critical nitrification step in the global nitrogen cycle across diverse ecological niches 4,5. Analysis of the available AOA genomes has revealed an abundance of copper-dependent proteins, which notably include multiple putative electron carrier proteins 4,5. Our research goals are to characterize the functional significance of the multiple, paralogous copper-dependent electron carrier proteins on the basis of their midpoint potentials and protein structure. This project complements work in other research labs at BGSU, and we have expanded involvement to include contributions from students in our undergraduate biochemistry laboratory course. Specifically, we have incorporated a research component into the curriculum where students apply concepts and techniques to purify previously-unstudied blue-copper proteins from AOA 6. We strive to enhance student engagement and strengthen scholarly activities such as experimental design, data analysis and communication of scientific results while simultaneously advancing our research objectives.

Creating Simple Tools To Advance Citizen-driven Investigations In The Environment

Our final research area is a collaborative venture involving BGSU faculty in three departments, including fellow Center for Photochemical Sciences member Alexis Ostrowski. We have been developing simple tools to facilitate education and participation in environmental stewardship initiatives by students and members of the broader public. This project took shape in 2014 when the domestic water supply in Toledo was shut off to more than 400,000 residents due to contamination by toxins from a harmful algal bloom in Lake Erie. Throughout the world, there is growing interest for engaging students and members of the public to participate in environmental water quality testing, however there are challenges in providing non-experts with the means to collect, share and interpret reliable scientific data. With contributions from colleagues and students in the Chemistry, Computer Science and Visual Communications Technology departments, we have developed software and device technologies that allow users to perform spectrophotometric measurements with color-based water quality test kits using their smartphones 7. Geo-tagged data collected in the field can be easily shared as part of an engaging “workflow” to support citizen-driven water quality initiatives. Similar to the above, students in three courses at BGSU have been involved in testing and development of software and device prototypes. You can read more about this on the website for our project we call GeoGraph.

Primary Research Techniques

We employ a variety of techniques to achieve our research goals. X-ray crystallography is our primary method to obtain structural information of Fe-S cluster proteins bound to their cognate Fe-S clusters. Atomic level detail is critical for analyzing the effects of site-specific mutations on Fe-S cluster properties, and we have an established record of research involving preparation, crystallization and structure determination of macromolecules, including proteins harboring oxygen-sensitive ISCs. We are comparing wild-type and mutant protein constructs using assays to measure nascent Fe-S cluster stability and transfer to apoproteins, as well as with structural comparisons by X-ray crystallography. Fe-S protein characterization is accomplished using UV-Vis, circular dichroism and solution biophysical methods including light scattering and isothermal titration calorimetry (ITC) for protein:protein and protein:ligand interactions. Electrochemical analysis of redox-active Fe-S clusters bound in proteins is achieved through collaboration. We also use standard molecular biology and microbiology methods for bacterial overexpression and growth fitness trials. Undergraduate and graduate students interested in our research are welcome to contact Dr. Torelli directly.