Heptad insertion extends the E. coli ROP protein by 50.

Area of Science:

• Protein Engineering and Structural Biology
• Biophysics and Molecular Imaging

Background:

• Antibody flexibility complicates structural studies and functional measurements.
• A rigid bivalent ligand construct could reduce antibody flexibility for applications.
• Coiled-coil protein bundles offer a tunable scaffold for creating rigid constructs.

Purpose of the Study:

• To develop a rigid, extended protein module for controlling antibody flexibility.
• To engineer and characterize a lengthened coiled-coil protein for metrology applications.
• To assess the potential of this construct for antibody imaging and functional modulation.

Main Methods:

• A heptad-insertion heuristic was used to extend the E. coli ROP/ROM protein.
• Tryptophan and phenylalanine residues were introduced for enhanced quantitation and structural analysis.
• X-ray crystallography was employed to determine the structures of the engineered variants.

Main Results:

• A tryptophan mutant and a lengthened variant (6.5 nm) of the ROP/ROM protein were successfully engineered.
• The extended variant is approximately 150% of the native protein's length (4.3 nm).
• The crystal structures confirmed the successful extension and modification of the protein bundle.

Conclusions:

• Engineered coiled-coil protein bundles can serve as rigid modules for creating larger constructs.
• The lengthened ROP/ROM variant provides a stable, tunable scaffold for advanced applications.
• This protein module has potential applications in antibody metrology, imaging, and functional studies.