Three-state unfolding and self-association of maspin, a tumor-suppressing serpin.
Maspin is a tumor suppressor protein expressed by normal human mammary epithelium but not by many breast tumor cell lines. Recombinant human maspin (rMaspin) inhibits tumor cell motility, invasion, and metastasis and thus has potential value as an anti-cancer therapeutic. Maspin is a member of the serpin family and, although the molecular mechanism by which maspin acts is unknown, recent work suggests that tissue plasminogen activator is a potential target. A puzzling observation in previous cell culture studies was loss of rMaspin activity at higher protein concentrations. One hypothesis to explain these results is self-association of rMaspin at the higher concentrations, which would be consistent with the tendency of serpins to form noncovalent polymers. This hypothesis is addressed by examining the relationship between rMaspin stability and self-association. Urea denaturation of rMaspin at pH 7 and 25 degrees C and at protein concentrations ranging from 0.01 to 0.2 mg/ml has been monitored by circular dichroism and intrinsic tryptophan fluorescence. Denaturation profiles show a protein concentration dependence and indicate the presence of at least one unfolding intermediate. The results suggest that destabilization of native monomeric rMaspin leads to partial unfolding and formation of an intermediate which can self-associate.
Liu
T
T
LXR Biotechnology, Richmond, California 94804, USA.
Pemberton
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PA
Robertson
A D
AD
D002942:Circular Dichroism; D016147:Genes, Tumor Suppressor; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D015843:Serpins / Q000737:chemistry*; D014461:Ultracentrifugation; D014508:Urea / Q000737:chemistry
D011506:Proteins; D011994:Recombinant Proteins; C085344:SERPIN-B5; D015843:Serpins; D014508:Urea