Carbon monoxide binding by de novo heme proteins derived from designed combinatorial libraries

Area of Science:

• Biochemistry
• Protein Engineering
• Biophysics

Background:

• De novo heme proteins were created using a binary code strategy for 4-helix bundles.
• These proteins were not explicitly designed for heme or ligand binding, offering an unbiased functional assessment.
• Approximately half of the binary code proteins were previously found to bind heme.

Purpose of the Study:

• To assess the carbon monoxide (CO) binding capabilities of de novo heme proteins.
• To characterize CO binding affinity, kinetics, and the local heme environment.
• To establish a baseline for CO binding in engineered alpha-helical proteins.

Main Methods:

• Studied eight de novo heme proteins from combinatorial libraries.
• Measured CO binding affinity (dissociation constants, K(d)).
• Analyzed CO association kinetics (k(on)) and resonance Raman spectra of CO complexes.

Main Results:

• All eight proteins showed CO binding affinities in the low nanomolar range, comparable to myoglobin.
• CO association kinetics indicated a partially buried heme environment.
• Resonance Raman studies revealed a local environment around bound CO lacking hydrogen-bonding groups.

Conclusions:

• De novo heme proteins display CO binding properties similar to natural heme proteins like myoglobin.
• The heme environment in these proteins is partially buried and lacks hydrogen-bonding interactions with CO.
• These findings define a "default" CO binding range for alpha-helical proteins in the absence of specific design or evolutionary selection.