The introduction of non-canonical amino acids has been 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. The stability benefits of fluorine incorporation into proteins are known. However, structural requirements to accommodate these non-natural amino acids are not fully understood. It is also not known how the non-proteogenic nature of fluorine contributes to the thermodynamic parameters of enthalpy and entropy and whether they are similar to those of natural proteins. The research presented here aims to develop an understanding of how fluorination increases protein stability to inform future efforts of modulating proteins with non-canonical amino acids.
The de novo designed 4-helix bundle, α4, was used to study the stabilizing properties of hexafluoroleucine (hFLeu) incorporation. The stabilizing of α4 proteins is dependent on the position of hFLeu incorporation, with proteins incorporating hFLeu into all a or all d positions displaying the highest per hFLeu residue stability. This enhanced stability through optimized core packing is contrary to predictions of fluorine-fluorine contacts increasing stability via the “fluorous effect”.
X-ray crystal structure comparison of α4 proteins shows minimal protein structural perturbation from hFLeu due to the retention of hydrocarbon side chain shape. A lack of specific fluorous interactions indicates that stability-enhancing properties of hFLeu are better ascribed to hydrophobic volume. Comparing the stability of a highly fluorinated α4 protein, which contains nearly identical core volume to its non-fluorinated counterpart, confirms that fluorinated proteins enhance stability by a general increase in hydrophobicity. The protein compatibility of hFLeu is again confirmed through thermodynamic analysis of 12 α4 proteins. These proteins show a general trend of increased free energy of unfolding, entropy and heat capacity with increasing hydrophobic surface area, regardless of hydrocarbon or fluorocarbon nature.
Lastly, the NMR properties of fluorine make fluorinated amino acids useful for studying transient biological interactions. Binding of trifluoroethylglycine (tFeG) containing antimicrobial peptide, MSI-78, to lipids is investigated. The trifluoromethyl fluorine reporter displays sequence-dependent chemical shift and conformational mobility upon binding.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/94053/1/buerben_2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/94053/2/buerben_1.pd
