Native β-barrel substrates pass through two shared intermediates during folding on the BAM complex.

Area of Science:

• Molecular Biology
• Protein Folding
• Membrane Protein Assembly

Background:

• The β-barrel assembly machine (BAM) complex is crucial for folding β-barrel proteins into membranes.
• BAM in *Escherichia coli* processes a wide variety of substrates with different sizes and shapes.
• The mechanism by which BAM efficiently folds such diverse substrates remains unclear.

Purpose of the Study:

• To investigate the folding mechanism of β-barrel proteins by the BAM complex.
• To characterize stable intermediates during the in vivo folding of native β-barrel substrates.
• To understand how BAM accommodates diverse substrate structures.

Main Methods:

• Development of a novel disulfide crosslinking method to trap folding intermediates in vivo.
• Site-specific cysteine introduction into the BamA protein and substrate β-strands.
• Comparison of substrate strand residence times within the BamA lumen.

Main Results:

• The disulfide crosslinking method successfully trapped and characterized stable folding intermediates.
• Identical folding intermediate stages were observed for two structurally distinct native β-barrel substrates.
• These intermediates occur early in folding and just before completion.

Conclusions:

• Common folding barriers exist for different β-barrel substrates.
• The BAM complex likely addresses these shared challenges, enabling the efficient folding of diverse proteins.
• The identified intermediates provide insights into the catalytic mechanism of BAM.