The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers...

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• The Cro repressor from phage lambda exists as a dimer.
• A five-amino acid insertion can stabilize a monomeric form.
• Residue Phe58 is critical for the dimer-to-monomer transition.

Purpose of the Study:

• To investigate the structural and stability effects of a Phe58 to Tryptophan (F58W) mutation in the Cro repressor.
• To understand how this mutation impacts both monomeric and dimeric forms of Cro.
• To elucidate the structural basis for changes in DNA-binding affinity and protein stability.

Main Methods:

• X-ray crystallography was used to determine the structures of the Cro F58W mutant in both monomeric and dimeric states.
• Comparative structural analysis of the mutant forms against wild-type Cro and DNA-bound Cro.
• Analysis of protein stability and DNA-binding affinity.

Main Results:

• The F58W mutation stabilized both the monomer and dimer forms of Cro.
• The F58W monomer adopted a structure distinct from the original monomer, resembling non-DNA-bound wild-type Cro.
• The F58W dimer, while structurally similar to the native dimer, revealed conformational changes upon DNA binding and showed perturbed DNA-contact residues.
• The mutation enhanced thermal stability due to favorable solvent transfer free energy and reduced internal cavities in the dimer.

Conclusions:

• The F58W mutation provides a structural basis for understanding Cro repressor stability and DNA-binding modulation.
• Structural insights confirm significant conformational changes in Cro upon DNA binding.
• The study highlights the role of specific residues in protein quaternary structure and DNA interaction.