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.
Tan
Y J
YJ
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore. mcbtanyj@imcb.nus.edu.sg
Ting
A E
AE
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