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)
Sanyal
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Department of Pharmaceutical Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Marquis-Omer
D
D
Gress
J O
JO
Middaugh
C R
CR
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins / Q000737:chemistry*; D001692:Biological Transport; D016923:Cell Death; D002942:Circular Dichroism; D005098:Exotoxins / Q000737:chemistry*; D005454:Fluorescence Polarization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D016211:Transforming Growth Factor alpha / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D008081:Liposomes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D014364:Tryptophan; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A