Selection of potent inhibitors of farnesyl-protein transferase from a synthetic tetrapeptide combinatorial library.

Inhibitors of farnesyl-protein transferase (FPTase) show promise as anticancer agents. Based on the sequence of the protein substrates of FPTase (the CAAX sequence), potent and selective peptidomimetic inhibitors have been developed; these compounds share with the peptide substrate a free thiol and a C-terminal carboxylate. We have used a synthetic tetrapeptide combinatorial library to screen for new leads devoid of these features: the peptides were C-terminally amidated, and no free thiol was included in the combinatorial building blocks. To compensate for this negative bias, an expanded set of 68 amino acids was used, including both L and D as well as many non-coded residues. Sixteen individual tetrapeptides derived from the consensus were synthesized and tested; all were active, showing IC50 values ranging from low micromolar to low nanomolar. The most active peptide, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid (Ki = 2 nM), is also very selective showing little inhibitory activity against the related enzyme geranylgeranyl-protein transferase type I (IC50 > 50 microM). In contrast to CAAX-based peptidomimetics, D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma-carboxyglut amic acid appeared to mimic the isoprenoid substrate farnesyl diphosphate as determined by kinetic and physical measurements. D-Tryptophan-Dmethionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid was a competitive inhibitor of FPTase with respect to farnesyl diphosphate substrate and uncompetitive with respect to CAAX substrate. Furthermore, we demonstrated that FPTase undergoes ligand dependent conformational changes in its circular dichroism spectrum and that D-tryptophan-D-methionine-D-4-chlorophenylalanine-L-gamma- carboxyglutamic acid induced a conformational change identical to that observed with farnesyl diphosphate ligand.

Spectroscopic characterization of tick anticoagulant peptide.

Tick anticoagulant peptide (TAP) is a disulfide rich potent inhibitor of factor Xa. Although this peptide is of potential clinical utility, very little is known about its higher order structure. Therefore, the secondary structure of recombinant TAP (rTAP) has been examined by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Both techniques suggest that rTAP is rich in beta-sheet structure. Disulfide bonds play a significant role in the folding and structural stability of rTAP. This is apparent from the resistance of rTAP to fluorescence-detected unfolding by guanidinium chloride (Gdn-HCl), unless disulfides are first reduced. The protein's tryptophan and tyrosine residues exhibit greater solvent exposure upon reduction of the cystines as indicated by fluorescence spectra and second derivative UV spectroscopy. A considerable amount of beta-structure appears to be retained after reduction of disulfides, although the CD spectrum manifests an increased amount of disordered structure in the reduced peptide. While rTAP does not bind the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) at neutral or acidic pH, the reduced peptide binds TNS at pH 2.0 but not at pH 7.0. The secondary structure of the reduced peptide at pH 2 is, however, similar to that at pH 7 as judged by CD spectroscopy. The reduced form of rTAP at acidic pH thus resembles a molten globule-like state.

Evidence for an equilibrium intermediate in the folding-unfolding pathway of a transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein.

TP40 is a chimeric protein containing transforming growth factor-alpha (TGF-alpha) at the N-terminus and a Cys-->Ala mutant (PE40 delta Cys) of a 40,000-dalton segment (PE40) of Pseudomonas exotoxin (PE). The guanidine hydrochloride (Gdn-HCl)-induced unfolding of TP40 and PE40 delta Cys has been studied by tryptophan fluorescence, circular dichroism (CD), and high-performance size exclusion chromatography (HPSEC). The equilibrium unfolding of both proteins involves at least one intermediate (I). In the I state(s), which may be induced by 1.3-2.0 M Gdn-HCl, the tertiary structure is fully or partially collapsed as detected by tryptophan fluorescence and near-UV CD, but the protein largely retains the native secondary structure and a semicompact shape as judged by far-UV CD and HPSEC, respectively. Soluble aggregates of TP40 and PE40 delta Cys are observed in addition to monomers at these intermediate (but not at higher) Gdn-HCl concentrations, suggesting that self-association is possibly mediated by thermodynamically stable, partially unfolded I states. The kinetics of refolding of TP40 upon dilution of Gdn-HCl involve two or more phases. Re-formation of secondary structure occurs rapidly (t 1/2 < 10 s) as determined by CD and is followed by a biphasic refolding of the native tertiary structure as detected by changes in tryptophan fluorescence. The midpoint (Tm) of the thermal unfolding transition occurs at a lower temperature when measured by tryptophan fluorescence than when detected by DSC and CD. These data suggest that Gdn-HCl and temperature can induce conformation(s) of TP40 that are distinct from native (N) and unfolded (U) states.(ABSTRACT TRUNCATED AT 250 WORDS)

A transforming growth factor-alpha-Pseudomonas exotoxin hybrid protein undergoes pH-dependent conformational changes conducive to membrane interaction.

TP40 is a chimeric protein containing transforming growth factor alpha (TGF-alpha) at the N-terminus and a derivative of a 40,000-Da segment (PE40 delta cys) of Pseudomonas exotoxin (PE). PE40 delta cys contains domains Ib, II, and III of PE in which the cysteines are mutated to alanines. The rationale for inclusion of TGF-alpha is to provide TP40 with selective targeting toward cells expressing the epidermal growth factor receptor (EGFr) on their surface [Pastan, I., & FitzGerald, D. (1989) J. Biol. Chem. 264, 15157-15160]. Translocation across endosomal membranes is thought to be a required step for cytotoxic activity of PE. This step is presumably facilitated by the low pH in endosomes which induces exposure of a hydrophobic surface of the protein, which in turn becomes available to interact with and translocate across the membrane. We have employed the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) and the intrinsic tryptophan fluorophores of TP40 to investigate pH-induced changes in the tertiary structure of this protein. The pH dependence of TP40 interaction with liposomes also provided a model for studying protein-membrane interactions. TNS fluorescence was markedly enhanced in the presence of TP40 below pH 4 and to a lesser degree between pH 7 and 5. A progressive red shift of tryptophan fluorescence with decreasing pH was also seen with the approximate midpoint for this transition occurring around pH 3. Both observations suggest that acidic pH induces exposure of hydrophobic regions of TP40, making them accessible to solvent and TNS. No major alteration of the secondary structure was manifested in the far-UV CD spectrum of TP40 upon a reduction in pH from 7 to 2. Thus, the low-pH-induced structural change of TP40 appears to involve a subtle exposure of one or more hydrophobic surfaces without an extensive unfolding of the protein's secondary structure. In the presence of anionic liposomes, a low-pH-induced blue shift of the TP40 tryptophan fluorescence was observed, suggesting that interaction with liposomes also required the low-pH conformation of the protein. However, the midpoint of this fluorescence blue shift occurred at approximately pH 5, which is presumably closer to the physiological pH within endosomes. Neutral liposomes failed to induce these spectral changes in TP40, implying a lack of interaction with these lipids. At acidic pH values between 2 and 4, self-association of TP40 in solution was detected by equilibrium sedimentation and quasielastic light scattering measurements. This probably results from intermolecular interaction between exposed hydrophobic surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Stabilization of the FK506 binding protein by ligand binding.

Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed.

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.