Biosynthesis and Stability of Coiled‐Coil Peptides Containing (2S,4R)‐5,5,5‐Trifluoroleucine and (2S,4S)‐5,5,5‐Trifluoroleucine

Montclare, Jin Kim; Son, Soojin; Clark, Ginevra A.; Kumar, Krishna; Tirrell, David A.

doi:10.1002/cbic.200800164

Cited by 63 publications

(56 citation statements)

References 41 publications

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“…Self-association of the peptide juxtaposes the a and d positions and results in the formation of a hydrophobic core. Fluorinated Leu analogues have previously been incorporated into the d positions of A1; the resulting proteins exhibited improved resistance to thermal and chemical denaturation with minimal differences in secondary structure (9,11,12). In this work, the surface-exposed Asp residue at the f position of the third heptad (position 34) was replaced by Trp, which serves as a fluorescence probe (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

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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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“…Whereas increasing concentrations of ethanol or isopropanol caused the helices of the latter protein first to dissociate into monomeric helices and then to completely unfold, these solvents had little effect on the fluorinated protein. Introduction of one CF 3 group in leucine generates two diastereoisomeric 5,5,5-trifluoroleucines that have both been incorporated into recombinant peptides expressed in an E. coli strain [122]. Both coiled-coil homodimers of peptides bearing one or the other isomers showed increased thermal stability relative to the non-fluorinated dimers.…”

Section: Amino Acids and Proteins With Fluorinated Patchesmentioning

confidence: 99%

Highly fluorinated amphiphilic molecules and self-assemblies with biomedical potential

Riess¹

2009

Current Opinion in Colloid & Interface Science

124

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“…Thus, fluorination improved the helical structure for both C+TFL and Q+TFL by 3 and 6.4 fold, respectively (Figure 2a), consistent with previous studies. 26 −29 To determine the effect of fluorination on protein stability, the melting temperature (T m ) was obtained by monitoring the changes in ellipticity at 222 nm as a function of temperature. The C and Q proteins demonstrated similar stabilities with a T m of 42 and 39°C, respectively (Figure 2a, Table S2).…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Influence of Fluorination on Protein-Engineered Coiled-Coil Fibers

Zhang

Srivastava

et al. 2015

Biomacromolecules

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We describe the design and characterization of fluorinated coiled-coil proteins able to assemble into robust nano- and microfibers. Fluorination is achieved biosynthetically by residue-specific incorporation of 5,5,5-trifluoroleucine (TFL). The fluorinated proteins C+TFL and Q+TFL are highly α-helical as confirmed via circular dichroism (CD) and more resistant to thermal denaturation compared to their nonfluorinated counterparts, C and Q. The fluorinated proteins demonstrate enhanced fiber assembly at pH 8.0 with higher order structure in contrast to nonfluorinated proteins, which are unable to form fibers under the same conditions. Ionic strength dependent fiber assembly is observed for fluorinated as well as wild-type proteins in which the fluorinated proteins exhibited more stable, thicker fibers. The fluorinated and nonfluorinated proteins reveal metal ion-dependent small molecule recognition and supramolecular assemblies. In the presence of Zn (II), enhanced thermal stability and fiber assembly is observed for the fluorinated proteins and their nonfluorinated counterparts. Whereas Ni (II) promotes aggregation with no fiber assembly, the stabilization of α-helix by Zn (II) results in enhanced binding to curcumin by the fluorinated proteins. Surprisingly, the nonfluorinated proteins exhibit multiple-fold increase in curcumin binding in the presence of Zn (II). In the context of the growing number of protein-based fiber assemblies, these fluorinated coiled-coil proteins introduce a new paradigm in the development of highly stable, robust self-assembling fibers under more physiologically relevant pH conditions that promotes the binding and release of small molecules in response to external cues.

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Biosynthesis and Stability of Coiled‐Coil Peptides Containing (2S,4R)‐5,5,5‐Trifluoroleucine and (2S,4S)‐5,5,5‐Trifluoroleucine

Cited by 63 publications

References 41 publications

Hydration dynamics at fluorinated protein surfaces

Hydration dynamics at fluorinated protein surfaces

Highly fluorinated amphiphilic molecules and self-assemblies with biomedical potential

Influence of Fluorination on Protein-Engineered Coiled-Coil Fibers

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