Fluorine in a Native Protein Environment—How the Spatial Demand and Polarity of Fluoroalkyl Groups Affect Protein Folding

Jäckel, Christian; Salwiczek, Mario; Koksch, Beate

doi:10.1002/anie.200504387

Cited by 143 publications

(145 citation statements)

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“…Fluorinated amino acids have drawn special attention (4-16) because of the unusual solubility properties of fluorinated hydrocarbons. Several independent studies have shown that fluorination of coiled-coil and helix-bundle proteins leads to enhanced stability with respect to thermal or chemical denaturation (6)(7)(8)(9)(10)(11)(12), an effect attributed to the hyper-hydrophobic and fluorophilic character of fluorinated amino acid side chains.…”

mentioning

confidence: 99%

Hydration dynamics at fluorinated protein surfaces

Kwon

Yoo

Othon

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Water-protein interactions dictate many processes crucial to protein function including folding, dynamics, interactions with other biomolecules, and enzymatic catalysis. Here we examine the effect of surface fluorination on water-protein interactions. Modification of designed coiled-coil proteins by incorporation of 5,5,5-trifluoroleucine or (4S)-2-amino-4-methylhexanoic acid enables systematic examination of the effects of side-chain volume and fluorination on solvation dynamics. Using ultrafast fluorescence spectroscopy, we find that fluorinated side chains exert electrostatic drag on neighboring water molecules, slowing water motion at the protein surface.fluorine | noncanonical amino acids | protein engineering | solvation dynamics | ultrafast hydration T he past decade has witnessed substantial expansion in the number and diversity of noncanonical amino acids that can be incorporated into recombinant proteins expressed in bacterial cells (1-3). Fluorinated amino acids have drawn special attention (4-16) because of the unusual solubility properties of fluorinated hydrocarbons. Several independent studies have shown that fluorination of coiled-coil and helix-bundle proteins leads to enhanced stability with respect to thermal or chemical denaturation (6-12), an effect attributed to the hyper-hydrophobic and fluorophilic character of fluorinated amino acid side chains.Although both classes of compounds are hydrophobic, hydrocarbons and fluorocarbons differ in important ways (17)(18)(19)(20)(21)(22). The high electronegativity of fluorine renders the C-F bond both strongly polar and weakly polarizable (17,21,22). The dipole associated with the C-F bond exerts strong inductive effects on neighboring bonds (23) and can form reasonably strong electrostatic interactions with ionic or polar groups when the two moieties are appropriately positioned. The hydrophobic character of fluorinated compounds has been described as "polar hydrophobicity (17)," and is believed to play important roles in organic and medicinal chemistry. Furthermore, the C-F bond is significantly longer than the C-H bond, and the calculated volume of the trifluoromethyl group is about twice that of a methyl group (20). The studies described here constitute an attempt to understand more fully the interaction of water with fluorinated molecular surfaces, and to provide a sound basis for the use of fluorinated amino acids in the engineering of proteins with unique and useful physical properties.The hydration layer adjacent to protein surfaces exhibits properties different from those of bulk water; the more rigid and denser structure of the hydration layer plays a crucial role in protein structure, folding, dynamics, and function (24-26). Elucidation of the dynamic features of this region, on the time scales of atomic and molecular motion, is essential in understanding protein hydration. In the past decade, the knowledge of hydration on protein surfaces has been extensively expanded by studying the dynamic properties of biological water for various pro...

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mentioning

confidence: 99%

Hydration dynamics at fluorinated protein surfaces

Kwon

Yoo

Othon

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Effect Of Hfleu and Tbala On Stability Of A 4 Proteinsmentioning

confidence: 99%

“…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.…”

Section: Effect Of Hfleu and Tbala On Stability Of A 4 Proteinsmentioning

confidence: 99%

“…[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.…”

Section: Introductionmentioning

confidence: 99%

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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

et al. 2012

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Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4-helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t-butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.

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De NovoDesign of Proteins

Jensen

2009

Peptide and Protein Design for Biopharmaceutical Applications

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Fluorine in a Native Protein Environment—How the Spatial Demand and Polarity of Fluoroalkyl Groups Affect Protein Folding

Cited by 143 publications

References 38 publications

Hydration dynamics at fluorinated protein surfaces

Hydration dynamics at fluorinated protein surfaces

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

De NovoDesign of Proteins

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