Interdomain interactions in the chimeric protein toxin sCD4(178)-PE40: a differential scanning calorimetry (DSC) study.
The thermal denaturation of the chimeric protein toxin known as sCD4(178)-PE40 (sCD4-PE40) was studied using differential scanning calorimetry (DSC). sCD4-PE40 consists of HIV-binding domains of the T-cell membrane protein known as CD4 and the cytotoxic domains of Pseudomonas exotoxin A (PE40). sCD4-PE40 undergoes two DSC transitions. An endothermic transition associated with unfolding of the CD4 and PE40 components occurs at approximately 46 degrees C in buffered saline at pH 6.5. An exothermic transition associated with precipitation of unfolded protein occurs at higher temperatures. Both transitions are irreversible. DSC studies of solutions of pH 5.0 to 9.5 indicate that sCD4-PE40 shows maximal thermal stability at around pH 6.5. Variable pH experiments are also presented on solutions of sCD4(183) and PE40 revealing how these components denature as independent structural entities. sCD4(183) denaturation occurs at significantly higher temperatures than does the CD4 component of sCD4-PE40. PE40 denaturation occurs at the same temperatures as sCD4-PE40. These results suggest that the native CD4 and PE40 components are independent and non-interacting entities in the chimeric sCD4-PE40 molecule and that unfolding of the less-stable PE40 component induces unfolding of the CD4 component. These destabilizing interdomain interactions of sCD4-PE40 are in contrast to the stabilizing interactions which apparently exist in wild-type Pseudomonas exotoxin A between its PE40 domains and the cell binding domain of the native toxin (analogous to the CD4 component in sCD4-PE40). Reasons are discussed why the type of interdomain interactions observed for sCD4-PE40 might be the norm for chimeric proteins.
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins / Q000737:chemistry*; D015704:CD4 Antigens / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography / Q000379:methods; D004355:Drug Stability; D005098:Exotoxins / Q000737:chemistry*; D006361:Heating; D000922:Immunotoxins / Q000737:chemistry*; D008970:Molecular Weight; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D015704:CD4 Antigens; D005098:Exotoxins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C057689:CD4-Pseudomonas toxin