Folding stability modulation of the villin headpiece helical subdomain by 4-fluorophenylalanine and 4-methylphenylalanine.

HP36, the helical subdomain of villin headpiece, contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, and Phe58). Hydrophobic effects and electrostatic interactions were shown to be the critical factors in stabilizing this core and the global structure. To assess the interactions among Phe47, Phe51, and Phe58 residues and investigate how they affect the folding stability, we implanted 4-fluorophenylalanine (Z) and 4-methylphenylalanine (X) into the hydrophobic core of HP36. We chemically synthesized HP36 and its seven variants including four single mutants whose Phe51 or Phe58 was replaced with Z or X, and three double mutants whose Phe51 and Phe58 were both substituted. Circular dichroism and nuclear magnetic resonance measurements show that the variants exhibit a native HP36 like fold, of which F51Z and three double mutants are more stable than the wild type. Molecular modeling provided detailed interaction energy within the phenylalanine residues, revealing that electrostatic interactions dominate the stability modulation upon the introduction of 4-fluorophenylalanine and 4-methylphenylalanine. Our results show that these two non-natural amino acids can successfully tune the interactions in a relatively compact hydrophobic core and the folding stability without inducing dramatic steric effects. Such an approach may be applied to other folded motifs or proteins.

Asymmetric synthesis of 3-azide-4-fluoro-l-phenylalanine.

The asymmetric synthesis of N-Fmoc-protected 3-azide-4-fluoro-l-phenylalanine as a photoactive phenylalanine analog has been achieved by Schöllkopf's alkylation.

Improved stability and half-life of fluorinated phosphotriesterase using Rosetta.

Recently we demonstrated that incorporating p-fluorophenylalanine (pFF) into phosphotriesterase dramatically improved folding, thereby leading to enhanced stability and function at elevated temperatures. To further improve the stability of the fluorinated enzyme, Rosetta was used to identify multiple potential stabilizing mutations. One such variant, pFF-F104A, exhibited enhanced activity at elevated temperature and maintained activity over many days in solution at room temperature.

Protonation-coupled redox reactions in planar antiaromatic meso-pentafluorophenyl-substituted o-phenylene-bridged annulated rosarins.

Proton-coupled electron transfer (PCET) processes are among the most important phenomena that control a variety of chemical and biological transformations. Although extensively studied in a variety of natural systems and discrete metal complexes, PCET mechanisms are less well codified in the case of purely organic compounds. Here we report that a planar ß,ß'-phenylene-bridged hexaphyrin (1.0.1.0.1.0), a 24 π-electron antiaromatic species termed rosarin, displays unique redox reactivity on protonation. Specifically, treatment with acid (for example, HI) yields a 26 π-electron aromatic triprotonated monocationic species that is produced spontaneously via an intermediate-but stable-25 π-electron non-aromatic triprotonated monoradical dication. This latter species is also produced on treatment of the original 24 π-electron antiaromatic starting material with HCl or HBr. The stepwise nature of the proton-coupled reduction observed in the planar rosarin stands in marked contrast to that seen for non-annulated rosarins and other ostensibly antiaromatic expanded porphyrinoids.

Characterization of antinociceptive potency of endomorphin-2 derivatives with unnatural amino acids in rats.

This study reports on the in vivo effects of four endomorphin-2 (EM-2) derivatives (EMD1-4) containing unnatural amino acids, i.e. 2-aminocyclohexanecarboxylic acid (Achc2), para-fluorophenylalanine (pFPhe4), ß-methylphenylalanine (ßMePhe4) and/or 2',6'-dimethyltyrosine (Dmt1). After induction of osteoarthritis by monosodium iodoacetate into the ankle joint of male Wistar rats, a chronic intrathecal catheter was inserted for spinal drug delivery. The mechanical threshold was assessed by a dynamic aesthesiometer. Intrathecal injection of the original EM-2 and the ligands (0.3-10 µg) caused dose-dependent antiallodynic effects. The comparison of the different substances revealed that EMD3 and EMD4 showed more prolonged antinociception than EM-2, and the effects of the highest dose of EMD4 were comparable to morphine, while EMD3 caused paralysis at this dose. The potency of the different ligands did not differ from EM-2. The results show that the derivatives of EM-2 have similar in vivo potency to the original ligand, but their effects were more prolonged suggesting that these structural modifications may play a role in the development of novel endomorphin analogues with increased therapeutic potential.

Modulating substrate specificity of histone acetyltransferase with unnatural amino acids.

Controlling the substrate specificity of enzymes is a major challenge for protein engineers. Here we explore the effects of residue-specific incorporation of ortho-, meta- and para-fluorophenylalanine (oFF, mFF, pFF) on the selectivity of human histone acetyltransferase (HAT) protein, p300/CBP associated factor (PCAF). Varying the position of the fluorine group in the phenylalanine ring confers different effects on the ability of PCAF to acetylate target histone H3 as well as non-histone p53. Surprisingly, pFF-PCAF exhibits an increase in activity for non-histone p53, while mFF-PCAF is selective for histone H3. These results suggest that global incorporation of unnatural amino acids may be used to re-engineer protein specificity.

Positional effects of monofluorinated phenylalanines on histone acetyltransferase stability and activity.

To explore the impact of global incorporation of fluorinated aromatic amino acids on protein function, we investigated the effects of three monofluorinated phenylalanine analogs para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the stability and enzymatic activity of the histone acetyltransferase (HAT), tGCN5. We selected this set of fluorinated amino acids because they bear the same size and overall polarity but alter in side chain shape and dipole direction. Our experiments showed that among three fluorinated amino acids, the global incorporation of pFF affords the smallest perturbation to the structure and function of tGCN5.

Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells.

Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC(50) values in the low micromolar range (3-15 microM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.

Phenylalanine side chain behavior of the intestinal fatty acid-binding protein: the effect of urea on backbone and side chain stability.

The equilibrium unfolding behavior of the intestinal fatty acid-binding protein has been investigated by (19)F-NMR after incorporation of 4-fluorophenylalanine and by pulsed field gradient diffusion (1)H-NMR. At low urea concentrations (0-3 m) but prior to the global unfolding that begins at 4 m urea, the protein exhibits dynamic motion in the backbone and an expanded hydrodynamic radius with no major change in the side chain orientation. As monitored by two-dimensional (19)F-(19)F nuclear Overhauser effect, the distance between two phenylalanine residues (Phe(68) and Phe(93)) located in the two different beta-sheets that enclose the internal cavity did not change up to 4 m urea. Additionally, the chemical shifts of these two residues changed almost identically as a function of denaturant. At all urea concentrations, as well as in the native protein, multiple conformations exist. These conformers interconvert at different rates under different conditions, ranging from slow exchange by showing separate peaks in the native state to intermediate exchange at intermediate urea concentrations. Residual structure persisted around Phe(62) even at very high concentrations of denaturant, suggesting that region as a nucleation site during folding. The results were compared with previous studies examining the backbone behavior (Hodsdon, M. E., and Frieden, C. (2001) Biochemistry 40, 732-742) and suggest that the side chains show more stability than the backbone prior to global unfolding of the protein.

Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR.

The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the C-terminal domain. To examine the folding properties of PapD, the protein was both uniformly and site-specifically labeled with p-fluoro-phenylalanine ((19)F-Phe) for (19)F NMR studies, in conjunction with those of circular dichroism and fluorescence. In equilibrium denaturation experiments monitored by (19)F NMR, the loss of (19)F-Phe native intensity for both the N- and C-terminal domains shows the same dependence on urea concentration. For the N-terminal domain the loss of native intensity is mirrored by the appearance of separate denatured resonances. For the C-terminal domain, which contains residues Phe 168 and Phe 205, intermediate as well as denatured resonances appear. These intermediate resonances persist at denaturant concentrations well beyond the loss of native resonance intensity and appear in kinetic refolding (19)F NMR experiments. In double-jump (19)F NMR experiments in which proline isomerization does not affect the refolding kinetics, the formation of domain-domain interactions is fast if the protein is denatured for only a short time. However, with increasing time of denaturation the native intensities of the N- and C-terminal domains decrease, and the denatured resonances of the N-terminal domain and the intermediate resonances of the C-terminal domain accumulate. The rate of loss of the N-terminal domain resonances is consistent with a cis to trans isomerization process, indicating that from an equilibrium denatured state the slow refolding of PapD is due to the trans to cis isomerization of one or both of the N-terminal cis proline residues. The data indicate that both the N- and C-terminal domains must fold into a native conformation prior to the formation of domain-domain interactions.

Virtual screening for binding of phenylalanine analogues to phenylalanyl-tRNA synthetase.

Although incorporation of nonnatural amino acids provides a powerful means of controlling protein structure and function, experimental investigations of amino acid analogues for utilization by the protein biosynthetic machinery can be costly and time-consuming. In this paper, we describe a computational protocol (HierDock) for predicting the relative energies of binding of phenylalanine analogues to phenylalanyl-tRNA synthetase (PheRS). Starting with the crystal structure of Thermus thermophilus PheRS without bound ligand, HierDock predicts the binding site of phenylalanine (Phe) within 1.1 A of that revealed by the crystal structure of PheRS cocrystallized with Phe. The calculated binding energies of Phe analogues in PheRS, using HierDock, correlate well with the translational activities of the same analogues in Escherichia coli. HierDock identifies p-fluorophenylalanine and 3-thienylalanine as especially good substrates for PheRS, in agreement with experiment. These results suggest that the HierDock protocol may be useful for virtual screening of amino acid analogues prior to experiment.

Fluorescence and 19F NMR evidence that phenylalanine, 3-L-fluorophenylalanine and 4-L-fluorophenylalanine bind to the L-leucine specific receptor of Escherichia coli.

The binding capacity of the L-leucine receptor from Escherichia coli was measured with L-phenylalanine and 4-fluoro-L-phenylalanine as substrates by fluorescence. The apparent dissociation constants (KD) for L-leucine, L-phenylalanine, and 4-fluoro-L-phenylalanine are 0.40, 0.18, and 0.26 respectively. 19F NMR data show protein-induced shifts for the 4-fluoro-L-phenylalanine peak and 3-fluoro-L-phenylalanine when receptor is present. Evidence points to the binding of only the L-isomers of these fluorine analogs.

Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli.

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.

The contribution of halogen atoms to protein-ligand interactions.

The three-dimensional structure of para-fluoro-D-phenylalanine (PFF) in its complex with the zinc protease carboxypeptidase A (CPA) has been determined at 2.0 A resolution by X-ray crystallographic methods. The structure reveals that the para-fluorobenzyl side chain of the inhibitor is buried in the S'1 hydrophobic pocket of the enzyme. Intriguingly, this ligand molecule inhibits CPA better than its amino acid analogues D-phenylalanine (D-Phe) and D-tyrosine (D-Tyr) by factors of 4 and 5, respectively. Moreover, the para-fluoro derivative is a better inhibitor than para-chloro- or para-bromo-D-phenylalanine by nearly a factor of 50. This result is consistent with binding enhancements realized in other protein complexes involving halogenated ligand molecules, regardless of whether the carbon-halogen group of the ligand makes specific polar interactions or non-specific hydrophobic interactions with its protein host. In the CPA-PFF complex, the fluorine atom of PFF does not make any direct polar contact with the enzyme, and the contact surface area of the protein-ligand interface is only slightly greater, although more hydrophobic, than that of D-Phe and D-Tyr. Therefore, we conclude that the slight binding enhancement measured for PFF relative to D-Phe and D-Tyr arises predominantly from increasing the hydrophobic character of the protein-ligand interface, and not solely from increasing the degree of protein-ligand contact.