Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Folding

Background:

• Protein folding is essential for cellular function.
• Cofactors are present in over 30% of proteins, but their role in co-translational folding is unclear.
• Flavodoxin utilizes flavin mononucleotide (FMN) as a cofactor.

Purpose of the Study:

• To investigate the impact of flavin mononucleotide (FMN) on the co-translational folding of flavodoxin.
• To determine when FMN binding occurs during flavodoxin synthesis and folding.

Main Methods:

• Generation of ribosome-arrested nascent chains of flavodoxin.
• Analysis of C-terminally truncated protein segments.
• Utilizing Escherichia coli (strain BL21(DE3) Δtig::kan) for experiments.

Main Results:

• Flavin mononucleotide (FMN) is a limiting factor for flavoprotein saturation in E. coli.
• Apoflavodoxin cannot bind FMN while associated with the ribosome due to ribosome-induced folding effects.
• Cofactor binding occurs after the complete synthesis and release of apoflavodoxin from the ribosome.
• Released apoflavodoxin folds natively and binds FMN.

Conclusions:

• Flavodoxin folding and FMN binding are post-translational events.
• Ribosome stalling prevents co-translational cofactor incorporation.
• The cellular production of flavodoxin concludes with cofactor binding to the released, folded protein.