Spectroscopic studies on Tb3+ binding to S-100a protein.
Direct binding assay and fluorescence studies revealed that S-100a protein binds 2 mol of Tb3+/mol of protein at pH 6.6. The protein binds Tb3+ much more tightly than Ca2+, and the upper limit of the observed Kd value for Tb3+ is 3.5 x 10(-6) M. The Tb3+-binding site on the protein must be close to a tyrosine residue, as indicated by fluorescence excitation and emission spectra, where energy transfer from tyrosine is noted. Addition of Tb3+ resulted in a conformational change in the protein, as revealed by u.v.-difference spectroscopy and c.d. studies. Far-u.v. c.d. studies indicated the helical content to decrease from approx. 39% to 35% in the presence of Tb3+. From u.v.-difference-spectroscopy results the single tryptophan and the tyrosine chromophores in S-100a protein are blue-shifted (i.e. exposed to the solvent) in the presence of Tb3+ and the observed conformational changes are similar to those induced by Ca2+, suggesting that Tb3+ can be employed as a Ca2+ analogue in spectral studies with S-100a protein.
Mani
R S
RS
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Kay
C M
CM
D000818:Animals; D015415:Biomarkers; D002942:Circular Dichroism; D009418:S100 Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013725:Terbium / Q000378:metabolism*
D015415:Biomarkers; C030564:S-100 calcium-binding protein alpha subunit; D009418:S100 Proteins; D013725:Terbium