Solution characterization of the dynamic conjugative entry exclusion protein TraG

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• The R100 plasmid encodes a type IV secretion system crucial for horizontal gene transfer in bacteria.
• This system contributes significantly to the spread of antibiotic resistance genes among bacterial pathogens.
• The TraG protein, specifically its periplasmic domain (TraG*), plays a key role in regulating DNA transfer and preventing redundancy.

Purpose of the Study:

• To investigate the structural dynamics and stability of the TraG* protein from the R100 plasmid.
• To elucidate the role of the N-terminal region of TraG* in its overall structure and function.
• To understand the molecular mechanisms underlying entry exclusion mediated by TraG*.

Main Methods:

• Thermofluor assays to assess protein stability.
• Circular dichroism spectroscopy to analyze protein secondary structure.
• Collision-induced unfolding-mass spectrometry (CIU-MS) for probing protein unfolding pathways.
• Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) and small-angle X-ray scattering (SAXS) for structural characterization.

Main Results:

• Structural studies revealed a dynamic region between the N- and C-terminal domains of TraG*.
• An N-terminal truncation mutant of TraG* exhibited increased stability and reduced disordered content compared to the full-length protein.
• The N-terminal 45 residues of TraG* are proposed to function as a flexible linker region.

Conclusions:

• The N-terminal residues of TraG* are critical for its conformational flexibility and likely act as a linker.
• Understanding TraG* structure-function relationships can inform strategies to combat the spread of antibiotic resistance.
• The dynamic nature of TraG* is integral to its role in preventing redundant DNA transfer during bacterial conjugation.