Synthesis, physicochemical characterization, and crystallization of a putative retro-coiled coil

Area of Science:

• Protein Chemistry
• Structural Biology
• Biophysics

Background:

• Artificial HIV enhancer-binding polypeptides can be dimerized using leucine zipper motifs.
• Previous attempts to dimerize peptides using retro sequences of leucine zippers were not fully explored.
• Retro proteins offer potential for novel structural and functional applications.

Purpose of the Study:

• To synthesize and characterize a retro version of a leucine zipper (r-LZ35) as a precursor for fusion proteins.
• To create a disulfide-crosslinked retro-leucine zipper peptide (r-LZ38)2.
• To investigate the structural properties and dimerization behavior of the retro-leucine zipper.

Main Methods:

• Synthesis of the retro-leucine zipper peptide (r-LZ35).
• N-terminal extension and disulfide bond formation to create (r-LZ38)2.
• Physicochemical characterization using circular dichroism and sedimentation equilibrium studies.

Main Results:

• The retro peptide r-LZ35 exists as an unstructured monomer at low concentrations but forms helical structures and tetramers at higher concentrations.
• The disulfide-crosslinked peptide (r-LZ38)2 forms a stable, noncovalent, helical dimer even at low micromolar concentrations.
• Crystallization of (r-LZ38)2 has been achieved, with X-ray structural analysis pending.

Conclusions:

• The disulfide-crosslinked retro-leucine zipper (r-LZ38)2 demonstrates stable helical dimerization, suitable for protein structural studies and design.
• This work validates the structural and functional potential of retro proteins.
• The findings pave the way for developing retro-leucine zipper fusion proteins.