Fluorinated proteins: from design and synthesis to structure and stability

Area of Science:

• Biochemistry
• Protein Engineering
• Fluorine Chemistry

Background:

• Fluorine's unique properties are valuable in modulating biological molecules.
• Perfluorinated molecules exhibit extreme stability and phase segregation (fluorous effect).
• The fluorous effect is utilized in organic synthesis for purification.

Purpose of the Study:

• To explore engineering perfluorocarbon properties into proteins.
• To investigate the effects of incorporating a fluorinated amino acid (hexafluoroleucine) into a model protein (α4H).
• To understand the basis of fluorinated side chain stabilization in proteins.

Main Methods:

• Synthesis of hexafluoroleucine, a fluorinated amino acid.
• Design and construction of a model 4-helix bundle protein (α4H) with a leucine-packed hydrophobic core.
• Repacking the hydrophobic core of α4H with combinations of leucine and hexafluoroleucine.
• Thermodynamic and structural studies (crystal structures, unfolding measurements) of wild-type and fluorinated proteins.

Main Results:

• Fluorination enhances protein stability against chemical, thermal, and proteolytic denaturation.
• No evidence for special fluorine-fluorine interactions mediating protein-protein interactions.
• Crystal structures show fluorinated residues fit the hydrophobic core with minimal structural perturbation.
• Increased thermodynamic stability is attributed to increased buried hydrophobic surface area.

Conclusions:

• Fluorination is a general strategy for enhancing protein stability.
• Protein stabilization by fluorinated side chains is primarily due to the hydrophobic effect.
• Fluorinated amino acids can increase protein stability without altering native protein-protein interaction mechanisms.