Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Abstract

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4-helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t-butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.

Unexpected trends in the hydrophobicity of fluorinated amino acids reflect competing changes in polarity and conformation

“…[9][10][11][12][13] Simultaneously, minimizing changes in solute-solute packing upon mutations is desirable to ensure that protein structureand thus function -is preserved. 14,15 This is, however, difficult with the limited pool of canonical hydrophobic amino acids because their side chains differ in structure and volume. Fluorinated versions of those amino acids, i.e.…”

What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein

“…Addressing this question is of high importance as the fluorine-labelled protein variant, and not the wild type, is used in in cellula NMR spectroscopy to report on the native structural and dynamical features of proteins in vivo. In this context, it has been shown that extensively fluorinated amino acids can be particularly effective in increasing protein stability 28 due to the increase in buried hydrophobic surface area as identified in structures solved by X-ray crystallography 29 . Kitevski-LeBlanc and co-workers have shown that 19 F enrichment in fluoro-phenyalanine labelled calmodulin results in an increasing protein disorder which can be diminished by a decreased level of fluorination 30 .…”

(2S,4R)- and (2S,4S)-Perfluoro-tert-butyl 4-Hydroxyproline: Two Conformationally Distinct Proline Amino Acids for Sensitive Application in 19F NMR

“… 35 − 37 In contrast, in the most widely used direct analogue of a canonical α-amino acid, hexafluoroleucine (6 fluorines), the two trifluoromethyl groups are diastereotopic, exhibiting separate resonances, and each is coupled to the methine hydrogen, resulting in reduced peak intensity due to each trifluoromethyl group existing as a doublet (four total peaks observed). 18 , 38 − 41 …”