Structural guidelines for stabilization of α-helical coiled coils via PEG stapling.

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Peptide and protein macrocyclization (stapling) enhances conformational stability and target binding.
• Limited guidelines exist for optimal interhelical staple placement and length in coiled coils.
• Rational design of stapled peptides requires better understanding of staple impact.

Purpose of the Study:

• To explore the impact of interhelical polyethylene glycol (PEG) stapling on coiled coil stability and proteolytic resistance.
• To develop structure- and sequence-based guidelines for optimal PEG staple incorporation.
• To demonstrate rational design of stabilized PEG-stapled proteins.

Main Methods:

• Investigated PEG stapling in a model disulfide-bound heterodimeric coiled coil.
• Analyzed the effect of staple location relative to a disulfide crosslink.
• Assessed different staple positions (e/g', f/b', b/c') and varying PEG staple lengths.
• Evaluated PEG linker variations and conjugation methods.

Main Results:

• Staples are more stabilizing farther from existing disulfide crosslinks.
• Specific staple positions (e/g') showed greater stabilization than others (f/b', b/c').
• Optimal PEG staple lengths vary depending on the staple position.
• PEG stapling demonstrates tolerance to linker variations and conjugation modes.
• Developed guidelines enabled rational design of a stabilized HER-2 affibody.

Conclusions:

• Provides crucial guidelines for rational design of PEG-stapled coiled coils.
• Optimized PEG stapling significantly enhances protein conformational stability and proteolytic resistance.
• Demonstrates successful application in designing a stabilized HER-2 affibody for potential therapeutic applications.