Research Summary: We characterized TtfA, a previously uncharacterized membrane-anchored, DNA-binding protein from Mycobacterium tuberculosis, revealing its structural stability, membrane localization, and potential role in DNA organization.
Author interview
Saksham Jain is currently a PhD student in the Department of Bioengineering, IISc. The work for this publication was pursued during his BS-MS at IISER Bhopal under a jointly guided project by Prof Vikas and Prof Mahalakshmi.
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Lab: Dr. Vikas Jain & Dr. R. Mahalakshmi, Indian Institute of Science Education and Research (IISER) Bhopal
What was the core problem you aimed to solve with this research?
Despite the essentiality of membrane proteins in Mycobacterium tuberculosis (Mtb), most remain poorly characterized due to challenges in expression and purification. Our aim was to biochemically and structurally characterize TtfA, a membrane-associated protein implicated in trehalose monomycolate transport and DNA interaction.
How did you go about solving this problem?
In our previous study (Palande et al., ACS Infect. Dis. 2024), we performed LC-MS-based proteomic screening to identify outer membrane proteins in Mtb with potential roles in small molecule uptake. From this dataset, TtfA stood out due to its predicted domain architecture containing an alpha-beta mix profile. Also previous study failed to obtain an overexpressing Mtb strain of this protein. Based on these features, we selected TtfA for further structural and functional characterization. We expressed the protein in E. coli and M. smegmatis, confirmed its membrane localization via fluorescence microscopy, and assessed its secondary structure in detergent micelles using CD spectroscopy. Finally, we demonstrated its ability to bind DNA through electrophoretic mobility shift assays, supporting its potential role in nucleoid organization.
How would you explain your research outcomes (Key findings) to the non-scientific community?
We discovered the function of a previously unknown protein TtfA from Mtb, the bacterium causing tuberculosis. It can attach to the cell membrane and bind to DNA, much like histones do in our own cells. This may help the bacterium organize its genetic material and survive stress. Importantly, this protein is also unusually stable, making it a strong candidate for future antibiotic targeting.
What are the potential implications of your findings for the field and society?
Our study establishes TtfA as a highly stable, membrane-anchored DNA-binding protein in Mycobacterium tuberculosis, expanding its functional repertoire beyond previously hypothesized roles in trehalose monomycolate (TMM) transport. By resolving the predicted structural features—an N-terminal transmembrane helix, a central β-sheet flanked by α-helices, and an unstructured C-terminal tail—we reveal for the first time that TtfA’s extramembranous domains interact with DNA, suggesting a role in nucleoid organization. This redefines how we view membrane-associated proteins in Mtb—not merely as transporters but as regulators of genome architecture. Furthermore, the protein’s folding stability and sensitivity to pH and detergents provide a tractable system for developing in vitro assays to probe its biochemical functions and interactions. These insights could pave the way for discovering novel anti-TB strategies that target essential membrane-linked processes beyond classical drug targets.
What was the exciting moment during your research?
The most exciting moment came after months of failed attempts at purification—when I finally obtained pure, stable TtfA protein. Conventional Ni-NTA affinity purification wasn’t working, and my protein either washed off in the flowthrough or co-eluted with contaminants, turning dark and unstable. It was only after realizing that DNA contamination was co-eluting with the protein that I optimized my protocol to remove it. That first clean prep was an immense relief. Even more rewarding was discovering that the purified protein showed robust DNA-binding activity, which we confirmed using EMSA and other assays—validating both the hypothesis and the long effort.
Paper reference: Jain, S., Patil, A.G., Patil, S. et al. Mycobacterium tuberculosis TtfA is a Highly Stable Membrane-Anchored DNA-Binding Protein. J Membrane Biol (2025). https://doi.org/10.1007/s00232-025-00352-5
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