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The introduction of non-canonical amino acids, that is amino acids that are not naturally found in proteins, has proved a useful tool to modify the properties of proteins. In particular, highly fluorinated amino acids have shown much promise in stabilizing a variety of protein folds towards degradation by proteases and unfolding by heat and chemical denaturants. Our research aims to understand how highly fluorinated amino acids integrate in to the structures of proteins and how fluorine confers stability on proteins. These studies should inform future efforts to modulate protein function and stability with non-canonical amino acids.
For our studies we focus on a model de novo designed 4-helix bundle protein, α4, to assess the effects of incorporating fluorinated amino acids such as hexafluoroleucine (hFLeu) into the hydrophobic core. The stabilizing effect of hFLeu is dependent upon its position in the core of α4. Incorporating hFLeu in an alternating pattern with leucine gives the highest stabilizing effect on a per residue basis. Interestingly, this is the opposite of what would be expected if favorable fluorine-fluorine contacts were responsible for increasing stability through the “fluorous effect”. A detailed thermodynamic analysis of 12 different variants of α4shows that the increased thermal stability of fluorinated proteins is primarily due to entropic effects, consistent with the increased hydrophobicity of fluorinated amino acids.
We solved the first X-ray structures of highly fluorinated proteins, which reveal why fluorine is so effective in stabilizing protein structures. Fluorination increases the buried hydrophobic surface area while at the same time closely preserving the shape of the side-chain. This allows the fluorinated amino acid to fit into the protein with minimal perturbation to its structure.
NMR properties of fluorine also make fluorinated amino acids useful for studying transient biological interactions. The antimicrobial peptide MSI-78 is a good model system for studying interactions of bioactive peptides with membranes. By incorporating trifluoroethylglycine, as a small and sensitive 19F NMR probe, at different positions in MSI-78 peptides we can study how the local structure and dynamics of the peptide change when it binds to the lipid bilayer. The fluorinated MSI-78 analogues exhibited position-specific changes in 19F chemical shift ranging from 1.28 to −1.35 ppm upon binding to lipid bicelles. Relaxation measurements show that the hydrophobic core of peptide–membrane complex undergoes the greatest decrease in mobility upon binding of the lipid bilayer, whereas residues that interact with lipid headgroups are more mobile. Our results provide support for the proposed mechanism of membrane disruption by MSI-78 and reveal new details about the dynamic changes that accompany membrane binding.
Publications
B.C. Buer, B.L. Levin and E.N.G. Marsh (2012). “Influence of Fluorination on the Thermodynamics of Protein Folding”. J. Am. Chem. Soc, 134, 13027 - 13034
Y. Suzuki, B.C. Buer, H.M. Al Hashimi and E.N.G. Marsh (2011). “Using Fluorine NMR to Probe Changes in Structure and Dynamics of Membrane-Active Peptides Interacting with the Lipid Bilayer”. Biochemistry, 50, 5979–5987
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