STU BORMAN, C&EN WASHINGTON

Replacing hydrophobic amino acid residues in peptides with polyfluorinated amino acid derivatives can cause profound increases in peptide stability, two research groups have found. The studies could have important implications for the design of novel enzymes and therapeutic proteins where stability is a key design issue.

FLUORINATED GCN4 Kumar and coworkers created GCN4 peptides containing four trifluoroleucines and three trifluorovalines. GCN4 dimer is shown in both side (left) and top views. A total of 14 trifluoromethyl groups are buried in the hydrophobic core. Gray = carbon, red = oxygen, blue = nitrogen, white = hydrogen, yellow = sulfur, green = fluorine. Most amino acid side chains have been omitted for clarity.

One of the two groups discovered little change in conformation but a major increase in stability when four leucines in each of two peptides in the dimeric yeast transcription factor GCN4 were changed to trifluoroleucines. That work was carried out by chemistry professors William A. Goddard III and David A. Tirrell and coworkers Yi Tang, Nagarajan Vaidehi, Jeremy Kua, and Daniel T. Mainz of California Institute of Technology, along with biochemistry and biophysics professor William F. DeGrado and postdoc Giovanna Ghirlanda of the University of Pennsylvania [Biochemistry, 40, 2790 (2001)].

Tirrell and coworkers selected trifluoroleucine as a derivative because it is approximately the same size and shape as leucine and can be readily incorporated into recombinant proteins produced by genetically engineered microorganisms.

The researchers found that fluorination of only four residues of each 33-residue monomer of the dimeric GCN4 peptide results in a structure that is more resistant to thermal and denaturant-induced unfolding than the wild-type peptide. For example, the fluorinated dimer is stable up to 61 °C, whereas the natural dimer denatures at 48 °C. Yet the affinity and specificity of binding of the fluorinated dimer to its natural DNA targets remains essentially the same as in the native one. It's not yet clear what factors give rise to the enhanced stability, but the researchers are currently trying to obtain thermodynamic data on that. 

MEANWHILE, assistant professor of chemistry Krishna Kumar and graduate students Basar Z. Bilgiçer and Alfio Fichera of Tufts University have found that changing four leucines and three valines to fluorinated versions in a related GCN4-derived peptide has a similarly profound impact on stability of the GCN4 dimer [J. Am. Chem. Soc., 123, 4393 (2001)].

The immiscibility of fluorous phases in water and many organic solvents, Kumar and coworkers speculated, would provide sufficient driving force for fluorinated amino acid side chains of the dimer to collapse into a fluorous core--which appears to be what happened. The thermal stability of the dimer is increased by 15 °C and the unfolding free energy by about 1.0 kcal per mole. The conformation of the fluorous peptide derivative is once again similar to that of the native one.

Asked to comment on the findings, professor of chemistry and biochemistry Ronald T. Raines of the University of Wisconsin, Madison, credits earlier studies by research chemist István T. Horváth of Exxon Research & Engineering, Annandale, N.J.; chemistry professor Dennis P. Curran of the University of Pittsburgh; and others for demonstrating "the utility of fluorocarbons in synthetic chemistry." The two new studies benefit from that work but go on to show "that fluorinated amino acid residues are also a powerful weapon for protein engineers," he says.

FIRST EXAMPLE Tirrell and coworkers synthesized this dimeric GCN4 peptide substituted with four trifluoroleucine groups. Van der Waals radii of fluorines are shown as yellow spheres.

Raines and coworkers themselves recently demonstrated the stereoelectronic use of fluorination to stabilize the protein collagen [J. Am Chem. Soc., 123, 777 (2001)]. "The word 'stereoelectronic' is critical," Raines says, "because the conformational stability of our fluorous collagen does not arise from fluorocarbon-fluorocarbon affinity. Rather, it arises from another consequence of fluorine--its electronegativity. In our JACS paper, we showed that the stereochemistry of the fluorine atom is critical: 4R-fluoroproline greatly stabilizes collagen, whereas 4S-fluoroproline greatly destablizes collagen. Such stereoelectronic considerations will be important for many future experiments on fluorous proteins."

Chemistry professor Samuel H. Gellman, also at the University of Wisconsin, Madison, who specializes in protein design, says the Caltech-Penn and Tufts studies "represent significant achievements in a relatively new dimension of protein design that focuses on structures and functions lying beyond those we know from biology. In this type of work, the chemist asks not 'How do natural proteins work' but 'How can I extrapolate from the architectural principles inherent in natural proteins' "

In the two studies, Gellman says, "the extra-biological principle is the strong self-affinity of fluorocarbon fragments. Both research groups hypothesized that the hydrophobic 'glue' in a natural protein dimer, which arises from hydrocarbon association, could be replaced by an even stronger fluorocarbon-fluorocarbon affinity. The results strongly support this design hypothesis. Their success points toward a number of exciting applications, including enhanced conformational stability of modified proteins and engineered complementarity between designed proteins."

Kumar points out that designed peptides with fluorous core structures could be of use as high-temperature catalysts, membrane-spanning peptides, and peptides with enhanced compatibility with organic solvents. "A very promising use of this approach could lie in the design of membrane proteins, where a binary patterning scheme with fluorocarbon-hydrocarbon surfaces is used to drive folding within the membrane," he says.

 FLUOROUS DERIVATIZATION also might "serve as a 'final push' toward higher stability after other methods, such as rational design and directed evolution, have achieved some initial stabilization," Tirrell and coworkers note. For example, "Peptides that rely on hydrophobic side chains to form channels in membranes may ... exhibit increased membrane or interpeptide association upon fluorination."

The technique could be applicable to the engineering of enzymes, signaling molecules, and protein ligands," they add, "and may prove to be of broad utility in the engineering of robust macromolecular assemblies."