The Fluorous Effect in Proteins: Properties of α₄F₆, a 4-α-Helix Bundle Protein with a Fluorocarbon Core

Abstract

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 4F 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 4H, in which the core is packed with leucine. alpha 4F 6 exhibits much greater resistance to proteolysis by either chymotrypsin or trypsin than alpha 4H and resists unfolding by organic solvents far better than alpha 4H. Whereas increasing concentrations of ethanol or 2-propanol cause the helices of the alpha 4H tetramer first to dissociate into monomeric helices and then to completely unfold, these solvents have little effect on the structure of alpha 4F 6. In contrast, increasing the concentrations of the fluorinated alcohol trifluoroethanol promotes dissociation of both alpha 4H and alpha 4F 6 to monomeric helices, whereas the secondary structure of both peptides remains intact. (19)F NMR experiments indicate that the two peptides can form mixed alpha-helical alpha 4F 6:alpha 4H bundles and thus do not exhibit the self-segregating behavior predicted by the fluorous effect. We conclude that the properties of alpha 4F 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.

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

“…Although they each contain the same number of hFLeu residues, a 4 F 3 (6-13) and a 4 F 3 (17)(18)(19)(20)(21)(22)(23)(24) are significantly less stable than a 4 F 3 a and a 4 F 3 d. These differences may be explained by vertical packing interactions of the ''b-e'' and ''c-g'' interfaces, the importance of which has been highlighted in studies on other antiparallel coiled-coil proteins. [38][39][40] The knobs-into-holes packing of ''b-e'' and ''c-g'' interfaces for a 4 F 3 a and a 4 F 3 d contain only like residues, in which Leu residues pack exclusively into the vertical hole created by two Leu residues of the adjacent helix and hFLeu residues follow a similar packing arrangement at the opposite interface.…”

“…21 The introduction of three hFLeu residues stabilizes the folding of a 4 F 3 d by -8.6 kcal mol À1 relative to a 4 H. For the present studies we initially synthesized three new a 4 variants: a 4 F 3 (6-13), which contains hFLeu in place of Leu at positions 6, 10, and 13; a 4 F 3 (17)(18)(19)(20)(21)(22)(23)(24), which contains hFLeu in place of Leu at positions 17, 20, and 24; and a 4 tbA 6 , which contains the leucine analog b-t-butylalanine in place of Leu at all 6 ''a'' and ''d'' positions.…”

“…We were unable to obtain welldiffracting crystals of a 4 F 3 (17)(18)(19)(20)(21)(22)(23)(24), precluding this protein from structural comparison. (Table II), with all residues being well resolved except the last residue of both the A and B chains.…”

“…[12][13][14] However, our laboratory and others have focused on introducing highly fluorinated analogs of residues such as valine, leucine, isoleucine, and phenylalanine into proteins as a means of enhancing stability. 12,[15][16][17][18][19][20][21][22][23] In most cases, these modified proteins display significantly increased stability toward unfolding by chemical denaturants and organic solvents, as well as increased resistance to proteolytic degradation. 20,[24][25][26] Currently, our understanding of how fluorination stabilizes protein folding is impeded by the lack of structures for proteins containing highly fluorinated amino acids.…”

“…12,[15][16][17][18][19][20][21][22][23] In most cases, these modified proteins display significantly increased stability toward unfolding by chemical denaturants and organic solvents, as well as increased resistance to proteolytic degradation. 20,[24][25][26] Currently, our understanding of how fluorination stabilizes protein folding is impeded by the lack of structures for proteins containing highly fluorinated amino acids. Gellman and coworkers recently determined both NMR and X-ray structures of chicken villin headpiece subdomain (cVHP) in which pentafluorophenylalanine (pFPhe) replaced one of three Phe residues in the core.…”