Maturation strategy influences expression levels and cofactor occupancy in Fe-S proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Iron-sulfur (Fe-S) clusters are essential cofactors for numerous biological processes.
• Recombinant protein production often suffers from low Fe-S cluster occupancy, hindering structural and functional studies.
• Efficient maturation of Fe-S proteins is critical for understanding their roles in fundamental biology.

Purpose of the Study:

• To systematically compare different in vivo and in vitro strategies for maturing [4Fe-4S] proteins.
• To evaluate the impact of iron-sulfur cluster biogenesis pathways on protein yield and cluster incorporation.
• To identify optimal methods for producing stable and functional Fe-S proteins, avoiding common pitfalls.

Main Methods:

• Comparison of protein production in BL21(DE3) cells with various maturation strategies.
• Inclusion of chemical and semi-enzymatic reconstitution methods.
• Co-expression with iron-sulfur cluster (isc) and sulfur formation (suf) operons.
• Utilizing a cell strain lacking IscR and an engineered 'SufFeScient' derivative.
• Testing the applicability of in vivo strategies to radical SAM proteins like ThnB.

Main Results:

• Co-expression of Fe-S biogenesis pathways significantly influences protein yield and cluster content.
• In vivo maturation promotes correct protein folding and structural stability by ensuring cluster presence.
• In vivo maturation effectively reduces the formation of Fe-S aggregates compared to chemical reconstitution.
• Successful extension of in vivo maturation to the radical SAM protein ThnB.

Conclusions:

• In vivo maturation strategies, particularly co-expression of biogenesis pathways, are superior to chemical reconstitution for producing Fe-S proteins.
• These methods enhance protein yield, stability, and reduce aggregation, facilitating structural and functional analysis.
• The findings provide critical insights into optimizing Fe-S protein production and highlight limitations of traditional in vitro approaches.