Highly divergent dihydrofolate reductases conserve complex folding mechanisms

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein folding mechanisms are crucial for biological function.
• Amino acid sequence identity can be low between homologous proteins from different species.
• Understanding conserved folding pathways can reveal evolutionary relationships.

Purpose of the Study:

• To test if protein folding mechanisms are more conserved than amino acid sequences.
• To compare the folding mechanisms of dihydrofolate reductases (DHFR) from human, E. coli, and L. casei.
• To investigate the role of kinetic intermediates and energy barriers in protein folding.

Main Methods:

• Utilized intrinsic tryptophan fluorescence and ANS binding to study protein unfolding and refolding kinetics.
• Monitored native state development via methotrexate or NADPH binding.
• Analyzed complex kinetic mechanisms involving partially folded states and multiple energy barriers.

Main Results:

• All three DHFR homologs exhibited complex folding mechanisms with parallel folding channels and two types of on-pathway kinetic intermediates.
• Despite sequence identities below 30%, conserved fast, intermediate, and slow-folding events were observed.
• Ligand binding (methotrexate and NADPH) coincided with the rate-limiting formation of the native ensemble.

Conclusions:

• Protein folding pathways are highly conserved among evolutionarily related proteins, even with divergent sequences.
• The study provides strong evidence that similar folding mechanisms underlie the same protein fold across species.
• Conserved folding events highlight the evolutionary importance of maintaining functional protein structures.