Non-ionic detergent affects the conformation of a functionally active mutant of Bcl-X(L).
We found that a mutant, Bcl-X(L)(F131V), which was previously reported to have impaired binding capacity, can bind to Bax almost as strongly as wild-type Bcl-X(L). In the absence of detergent, the Bcl-X(L)(F131V) mutant adopts the same conformation as wild-type Bcl-X(L), as determined by circular dichroism spectroscopy and size-exclusion chromatography. However, non-ionic detergent induces a conformational change in the Bcl-X(L)(F131V) mutant and causes it to lose Bax-binding capacity. Wild-type Bcl-X(L), on the other hand, is more resistant to detergent-induced effects and retains its ability to bind Bax in the presence of detergent. Since it has been shown that the Bcl-X(L)(F131V) mutant has nearly the same anti-apoptotic activity as wild-type Bcl-X(L), it would be likely that the Bcl-X(L)(F131V) mutant can adopt the wild-type conformation, rather than the detergent-induced conformational state and can bind to Bax in vivo. Therefore, our data demonstrated that non-ionic detergent can have unpredicted effects on protein conformation, differential effects on wild-type and mutant Bcl-X(L) proteins in this case and may cause complications in the interpretation of in vitro binding studies.
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore. mcbtanyj@imcb.nus.edu.sg
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Ting
D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D009154:Mutation; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494826:BAX protein, human; C494811:BCL2L1 protein, human; D003902:Detergents; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D005982:Glutathione Transferase