An engineered disulfide bond in dihydrofolate reductase

Area of Science:

• Biochemistry
• Structural Biology
• Enzyme Engineering

Background:

• Escherichia coli dihydrofolate reductase (EcDHFR) is a crucial enzyme in folate metabolism.
• Protein engineering allows for the modification of enzyme structure and function.
• Disulfide bonds can stabilize protein structures and influence folding pathways.

Purpose of the Study:

• To engineer a novel disulfide bond in EcDHFR by site-directed mutagenesis.
• To investigate the structural and functional consequences of this engineered disulfide bond.
• To assess the impact of the disulfide bond on enzyme stability and folding.

Main Methods:

• Oligonucleotide-directed mutagenesis to substitute proline-39 with cysteine.
• Expression of the mutant protein in E. coli cytosol.
• X-ray crystallographic analysis of the wild-type and mutant enzymes.
• In vitro oxidation to form the disulfide bond.
• Guanidine hydrochloride denaturation and urea-gradient polyacrylamide gel electrophoresis to assess stability and folding.

Main Results:

• A disulfide bond was successfully engineered between cysteine-39 and cysteine-85 in EcDHFR.
• X-ray crystallography revealed van der Waals contact in the reduced mutant and a disulfide bond in the oxidized mutant.
• The disulfide-cross-linked enzyme exhibited increased stability (1.8 kcal/mol) against guanidine hydrochloride-induced unfolding.
• No significant change in overall conformation was observed, but thermal motion details were altered.
• The cross-linked enzyme showed altered folding/unfolding pathways with intermediate formation.
• Thermal denaturation resistance was not improved by the disulfide bond.

Conclusions:

• Engineered disulfide bonds can enhance protein stability against chemical denaturation.
• Disulfide bond formation can significantly alter protein folding pathways without major conformational changes.
• The study provides insights into structure-function relationships and protein folding dynamics in EcDHFR.