The fluorous effect in proteins:: Properties of α4F6, a 4-α-helix bundle protein with a fluorocarbon core

To test the prediction that extensively fluorinated (fluorous) proteins should be more stable and exhibit novel self-segregating behavior, the properties of the de novo designed model 4-alpha-helix bundle protein, alpha F-4(6), in which the hydrophobic core is packed entirely with the extensively fluorinated amino acid L-5,5,5,5',5',5'-hexafluoroleucine, have been compared with its nonfluorinated counterpart, alpha H-4, in which the core is packed with leucine. alpha F-4(6) exhibits much greater resistance to proteolysis by either chymotrypsin or trypsin than alpha H-4 and resists unfolding by organic solvents far better than a4H. Whereas increasing concentrations of ethanol or 2-propanol cause the helices of the alpha H-4 tetramer first to dissociate into monomeric helices and then to completely unfold, these solvents have little effect on the structure of alpha F-4(6)center dot In contrast, increasing the concentrations of the fluorinated alcohol trifluoroethanol promotes dissociation of both alpha H-4 and alpha F-4(6) to monomeric helices, whereas the secondary structure of both peptides remains intact. F-19 NMR experiments indicate that the two peptides can form mixed alpha-helical alpha F-4(6):alpha H-4 bundles and thus do not exhibit the self-segregating behavior predicted by the fluorous effect. We conclude that the properties of alpha F-4(6) are best explained by the more hydrophobic nature of the hexafluoroleucine side chain, rather than the low solubility of fluorocarbons in hydrocarbon solvents that forms the basis of the fluorous effect.