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)
Gress
J O
JO
Department of Pharmaceutical Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Marquis-Omer
D
D
Middaugh
C R
CR
Sanyal
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G
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D002942:Circular Dichroism; D005098:Exotoxins / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D016211:Transforming Growth Factor alpha / Q000737:chemistry*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D019791:Guanidine; D006146:Guanidines; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A