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The snake venom protein echistatin is a potent inhibitor of platelet aggregation. The inhibitory properties of echistatin have been attributed to the Arg-Gly-Asp sequence at residues 24-26. In this paper, sequence-specific nuclear magnetic resonance assignments are presented for the proton resonances of echistatin in water. The single-chain protein contains 49 amino acids and 4 cystine bridges. All of the backbone amide, C{sub alpha}H, and side-chain resonances, except for the {eta}-NH of the arginines, have been assigned. The secondary structure of the protein was characterized from the pattern of nuclear Overhauser enhancements, from the identification of slowly exchanging amide protons, from {sup 3}J{sub c{alpha}H-NH} coupling constants, and from circular dichroism studies. The data suggest that the secondary structure consists of a type I {beta}-turn, a short {beta}-hairpin, and a short-, irregular, antiparallel {beta}-sheet and that the Arg-Gly-Asp sequence is in a flexible loop connecting two strands of the distorted antiparallel {beta}-sheet.

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)

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)

Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].

Calmodulin (CaM) fragments 1-77 (CaM 1-77) and 78-148 (CaM 78-148) were prepared by tryptic cleavage of CaM. CaM 78-148 exhibited Ca2+-dependent binding to mastoparan X, Polistes mastoparan, and melittin with apparent dissociation constants less than 0.2 microM as judged from changes in the fluorescence spectrum and anisotropy of the single tryptophan residue of each of these cationic, amphiphilic peptides. This interaction was accompanied by a large spectral blue shift of the peptide fluorescence spectrum. These findings are consistent with earlier results [Malencik, D.A., & Anderson, S.R. (1984) Biochemistry 23, 2420-2428] on the binding of mastoparan X to CaM fragment 72-148. The binding of the peptide to CaM 78-148 also caused a significant loss of the accessibility of the peptide tryptophan to the fluorescence quencher acrylamide. The CaM 78-148 induced effects on the fluorescence spectra and tryptophan accessibility of the peptides were most pronounced for mastoparan X, a peptide with tryptophan on the apolar face of the putative amphiphilic helix. The data were comparable with results from parallel experiments on the Ca2+-dependent interaction of these peptides with intact CaM. Difference circular dichroic spectra suggested that binding to CaM 78-148 was associated with the induction of considerable degrees of helicity in the amphiphilic peptides, which by themselves have predominantly random coil structures in aqueous solution. This finding is also reminiscent of the interaction of these peptides with intact CaM.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional helical wheel diagrams and calculations of mean hydrophobic moments show mastoparan, mastoparan X, and Polistes mastoparan to have all the properties expected for amphiphilic helices. Circular dichroic properties are consistent with a random form for these peptides in dilute aqueous solution, but greater than 50% helix is apparent when the peptides are dissolved in 70% trifluoroethanol/water mixtures (v/v) or when the peptides are bound to calmodulin. Changes in the fluorescence spectra, anisotropy, and accessibility of tryptophan whose indole side chain is on the apolar surface of the amphiphilic helix imply a significant role for the apolar surface in the binding of the mastoparans and another amphiphilic peptide, melittin, to calmodulin. These data provide a useful model for designing high-affinity synthetic peptide inhibitors of calmodulin.