Fluorinated Coiled-Coil Proteins Prepared In Vivo Display Enhanced Thermal and Chemical Stability

Abstract

Fluorination of the hydrophobic core of a coiled‐coil protein significantly improved its stability toward thermal and chemical denaturation. 5′,5′,5′‐Trifluoroleucine (2) was efficiently incorporated into a leucine‐zipper protein in place of leucine (1) during E. coli biosynthesis. The fluorinated variant maintained stable secondary and tertiary structures under conditions that caused denaturation of the “wild‐type” protein.

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

“…As a nonfluorinated comparison we have determined the structure of a 4 tbA 6 , which contains b-t-butylalanine (tBAla) at all ''a'' and ''d'' positions. This side-chain has a surface area intermediate between that of Leu and hFLeu, so that introducing 24 tBAla residues into the core of a 4 tbA 6 should result in a buried hydrophobic surface area nearly identical to that of either a 4 F 3 d ora 4 F 3 (6)(7)(8)(9)(10)(11)(12)(13). Therefore, any differences in structure and stability are expected to arise primarily from the difference in the shapes of the side-chains.…”

“…Crystallization of a 4 F 3 d, a 4 F 3 (6-13) and a 4 tbA 6 To investigate how the changes in thermodynamic stability are related to changes in structure we crystallized three of these proteins, a 4 F 3 d, a 4 F 3 (6)(7)(8)(9)(10)(11)(12)(13) and a 4 tbA 6 and determined their structures by Xray crystallography. 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.…”

“…[8][9][10][11] The substitution of fluorine for hydrogen is generally considered sterically nonperturbing and many ''lightly'' fluorinated amino acids can be processed by endogenous tRNA synthetases. [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.…”

“…[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.…”

“…a 4 F 3 (6)(7)(8)(9)(10)(11)(12)(13) contains the same number of hFLeu residues as a 4 F 3 a and a 4 F 3 d, but the packing is now altered so that hFleu is introduced at one ''a'' position (10) adjacent to two ''d'' positions (6 and 13); this allows interaction between hFLeu residues on adjacent ''a'' and ''d'' positions to be evaluated. As a nonfluorinated comparison we have determined the structure of a 4 tbA 6 , which contains b-t-butylalanine (tBAla) at all ''a'' and ''d'' positions.…”