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MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria
Published on: February 23, 2021
Overexpression, purification, and site-directed spin labeling of the Nramp metal transporter from Mycobacterium
Ian Reeve1, David Hummel, Nathan Nelson
1Department of Biological Chemistry, University of California School of Medicine, Davis, CA 95616, USA.
Abstract:
It has long been recognized that the pathogenicity of a broad range of intracellular parasites depends on the availability of transition metal ions, especially iron. Nramp1 (natural resistance-associated macrophage protein 1), a proton-coupled divalent metal ion transporter, has been identified as a controlling factor in the resistance or susceptibility to infection with a diverse range of intracellular pathogens such as Toxoplasma, Salmonella, Mycobacterium, and Leishmania. The role of divalent metal ion transport is even more compelling given the existence of Nramp homologs in several intracellular parasites, such as mycobacteria. We have confirmed the functional homology of the Nramp homologue from Mycobacterium leprae by using a yeast complementation assay for divalent cation uptake. To facilitate a concerted biochemical and structural analysis of this important class of transporters, the M. leprae Nramp was expressed in Escherichia coli. Dual affinity tags were engineered at the N and C termini to allow for isolation of full-length protein at >95% purity. Site-directed spin labeling of Cys-299 reveals a flexible hinge-like domain. A weak dipolar interaction is detected between the nitroxide and paramagnetic transition ions, indicating this position is approximately 19 A from the nearest high affinity binding site.
Insights
The study confirms functional homology of Mycobacterium leprae Nramp, a metal ion transporter crucial for controlling intracellular parasite infections. This research facilitates further biochemical and structural analysis of Nramp transporters.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Pathogenicity of intracellular parasites relies on transition metal ions like iron.
- Natural resistance-associated macrophage protein 1 (Nramp1) regulates susceptibility to pathogens (Toxoplasma, Salmonella, Mycobacterium, Leishmania).
- Nramp homologs exist in intracellular parasites, suggesting conserved roles in metal ion transport.
Purpose of the Study:
- To confirm the functional homology of the Nramp homologue from Mycobacterium leprae.
- To facilitate biochemical and structural analysis of M. leprae Nramp.
- To characterize the M. leprae Nramp transporter.
Main Methods:
- Yeast complementation assay for divalent cation uptake.
- Expression of M. leprae Nramp in Escherichia coli.
- Site-directed spin labeling of Cys-299 and electron paramagnetic resonance (EPR) spectroscopy.
Main Results:
- Functional homology of M. leprae Nramp was confirmed via yeast complementation.
- Full-length M. leprae Nramp was purified to >95% purity using dual affinity tags.
- Site-directed spin labeling identified a flexible hinge-like domain at Cys-299, approximately 19 Å from a high-affinity binding site.
Conclusions:
- The M. leprae Nramp homolog is functionally active and shares homology with other Nramp proteins.
- The purified M. leprae Nramp provides a basis for detailed structural and biochemical studies.
- Structural insights suggest a flexible domain near the metal ion binding site, crucial for transporter function.
