Evidence for partial secondary structure formation in the transition state for arc repressor refolding and...

Area of Science:

• Protein folding and biophysics
• Molecular biology
• Structural biology

Background:

• Arc repressor refolding and dimerization are coupled processes.
• Understanding the transition state of protein folding is crucial for deciphering folding pathways.

Purpose of the Study:

• To investigate the integrity of secondary structure in the transition state of Arc repressor refolding.
• To determine how mutations affect the stability and kinetics of Arc repressor folding and dimerization.

Main Methods:

• Site-directed mutagenesis (Ala --> Gly substitutions) at solvent-exposed residues.
• Analysis of equilibrium stability and refolding/unfolding kinetics.
• Determination of Phi(F) values to probe transition state structure.

Main Results:

• Most Ala --> Gly mutations in alpha-helices and beta-sheet destabilized the Arc repressor, primarily by reducing refolding rates.
• Some mutations indicated a lack of secondary structure at specific positions in the transition state.
• Phi(F) values for Ala --> Gly mutations were larger than for alanine substitutions, suggesting stronger backbone interactions in the transition state.
• Mutations affecting refolding rates clustered in alpha-helix A and B turns, potentially forming a folding nucleus.

Conclusions:

• The transition state of Arc repressor folding contains partially formed secondary structures.
• Secondary structure consolidation and hydrophobic core packing occur later in the folding pathway.
• Backbone interactions may play a more significant role than side chain interactions in the Arc repressor transition state.