Oxidative protein folding in bacteria project summary oxidative protein folding generates covalent linkages between the sulfur atoms of two cysteine residues, known as disulfide bonds (dsbs)

The National Institute of General Medical Sciences awarded the Trustees of Indiana University $415,822 on April 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to support fundamental research on oxidative protein folding in bacteria.

The research investigates disulfide bond formation in bacterial model systems, specifically addressing three key questions: the mechanisms enabling productive thiol oxidation when canonical disulfide-forming enzymes are inactive; the identity of proteins requiring disulfide bonds in complex cell envelopes; and substrate specificity among distinct thiol-disulfide oxidoreductases. The research employs bacterial genetics, chemical genetics, and biochemistry using two model organisms—Escherichia coli (gram-negative) and Mycobacterium smegmatis (acid-fast)—to dissect in vivo disulfide bond formation mechanisms and reveal novel oxidation pathways that assist protein folding.

Work is performed in Bloomington, Indiana, with a period of performance extending through February 28, 2031. The award is a Project Grant, a standard assistance type under NIGMS's broad portfolio of investigator-initiated biomedical research supporting basic science that advances understanding of biological processes and lays the foundation for disease diagnosis, treatment, and prevention advances.

Dsbs play a crucial role in the three-dimensional structure and biological function of exported proteins in prokaryotes and eukaryotes. Since the discovery of the first DSB catalyst in the bacterium escherichia coli, bacteria have been proven to be a powerful model system for understanding the molecular mechanisms that drive DSB formation. Our research aims to address three key questions in the field: 1) what principles enable productive thiol oxidation in essential proteins when canonical DSB-forming enzymes are inactive? 2) what proteins depend on dsbs in complex cell envelopes? And 3) do distinct thiol-disulfide oxidoreductases exhibit substrate specificity? My lab employs a multidisciplinary approach to dissect the fundamental mechanisms and consequences of DSB formation in vivo using bacteria. As model systems for our basic research program, we employ two model organisms, the gram-negative bacterium e. Coli and the acid-fast bacterium mycobacterium smegmatis, because (1) they each express one of the two divergent pathways known for DSB catalysis in prokaryotes, (2) they are genetically tractable with a plethora of tools developed over the past three decades, and (3) they have intricate cell envelopes thus many more proteins necessitate oxidative protein folding. The proposed research aims to leverage bacterial genetics, chemical genetics and biochemistry to advance the understanding of DSB formation in bacteria and reveal novel oxidation mechanisms assisting folding with canonical DSB-forming system, the contributions of dsbs in organisms with the alternative system and the determinants of substrate recognition and specificity among different classes of oxidoreductases. Ultimately, our work will inform how DSB forming pathways can be targeted for antibacterial innovation to combat diverse clinically relevant infections and harnessed to engineer proteins with enhanced stability. First, we will elucidate the mechanism of oxidation in essential proteins harboring dsbab systems. Preliminary TN- SEQ screens have already identified synthetic lethal interactions of a dsba mutant, which represent candidate genes that are likely involved. We will study these candidate genes through genetic and biochemical approaches. Second, we will characterize the DSB oxidation and isomerization systems in the mycobacterial cell envelope using genetic and biochemical approaches. We recently found that DSB formation is crucial for the folding of multiple essential proteins in mycobacteria. We aim to understand DSB formation in two different cell envelope architectures to dissect substrate preference between the two classes of dsba proteins capable of recognizing hundreds of substrates, through cysteine derivatization and enrichment proteomics. Collectively, our basic research will provide fundamental insights into novel oxidative reactions, the exported proteins that depend on dsbs, and the substrate specificity among thiol-disulfide oxidoreductases.