p53 unfolding detected by CD but not by tryptophan fluorescence.
Full-length human p53 protein was examined using tryptophan fluorescence and circular dichroism spectroscopy (CD) to monitor unfolding. No significant alteration in tryptophan fluorescence for the tetrameric protein was detectable over a wide range of either urea or guanidine hydrochloride concentrations, in contrast to results with the isolated DNA binding domain [Bullock et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14338]. Under similar denaturant conditions, CD demonstrated significant protein unfolding for the full-length wild-type protein, with increased apparent structure loss compared to that detected during thermal denaturation [Nichols and Matthews (2001) Biochemistry 40, 3847]. Examination of X-ray structures containing two of the four tryptophan residues of a p53 monomer suggested local environments consistent with quenched fluorophores. Exploration of p53 fluorescence using potassium iodide as a quencher confirmed that these fluorophores are already substantially quenched in the native structure, and this quenching is not relieved during protein unfolding.
Nichols
N M
NM
Department of Biochemistry & Cell Biology, Rice University, MS102, Houston, TX 77005, USA.
Matthews
K S
KS
D002942:Circular Dichroism; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D011193:Potassium Iodide / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*; D014508:Urea / Q000737:chemistry
D016159:Tumor Suppressor Protein p53; D011193:Potassium Iodide; D014364:Tryptophan; D014508:Urea; D019791:Guanidine