The quaternary structure of streptavidin in urea.
We report on the interactions of urea and guanidinium salts with streptavidin. Gel filtration chromatography in 0, 4, 6, and 7 M urea indicates that the streptavidin tetramer remains intact in urea. Biotin alters the electrophoretic mobility of streptavidin whether or not 6 M urea is present. The intrinsic fluorescence of streptavidin is increased and blue-shifted in 6 M urea. The fluorescence changes indicate the absence of unfolding. A conformational response to urea is possible, but much of the fluorescence change is due to urea binding as a weak biotin analog (Ka approximately 1.3 M-1). The resistance to structural perturbation by urea reflects the structural stability of streptavidin's anti-parallel beta-barrel motif. Unfolding is sluggish in 6 M guanidinium hydrochloride (half-time, approximately 50 days). After guanidinium thiocyanate unfolding, streptavidin can be refolded, but the unfolding and refolding transitions are centered at different concentrations of perturbant. Slow unfolding, with a 15th power dependence on guanidinium thiocyanate concentration, may be partially responsible for the noncoincidence of the unfolding and refolding processes. Nonequilibrium behavior is also seen in 6 M urea, as native streptavidin does not unfold and guanidinium thiocyanate unfolded streptavidin does not refold. Refolding does occur at lower concentrations of urea. Guanidinium thiocyanate only slowly unfolds the biotin-streptavidin complex. In the presence of biotin, unfolded streptavidin does not refold in 6 M guanidinium thiocyanate or in 6 M urea.
Kurzban
G P
GP
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Bayer
E A
EA
Wilchek
M
M
Horowitz
P M
PM
D001426:Bacterial Proteins / Q000737:chemistry*; D001710:Biotin / Q000737:chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019809:Streptavidin; D014508:Urea / Q000737:chemistry*
D001426:Bacterial Proteins; D006146:Guanidines; D001710:Biotin; D014508:Urea; D019809:Streptavidin; D019791:Guanidine