Mani R S, Kay C M
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochem J. 1987 Jun 15;244(3):559-63. doi: 10.1042/bj2440559.
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.
直接结合测定和荧光研究表明，在pH 6.6条件下，S-100a蛋白与每摩尔蛋白结合2摩尔Tb3+。该蛋白与Tb3+的结合比与Ca2+的结合紧密得多，观察到的Tb3+的Kd值上限为3.5×10(-6)M。如荧光激发和发射光谱所示，蛋白上的Tb3+结合位点一定靠近一个酪氨酸残基，在该光谱中可观察到从酪氨酸的能量转移。紫外差光谱和圆二色性研究表明，加入Tb3+会导致蛋白发生构象变化。远紫外圆二色性研究表明，在存在Tb3+的情况下，螺旋含量从约39%降至35%。根据紫外差光谱结果，在存在Tb3+的情况下，S-100a蛋白中的单个色氨酸和酪氨酸发色团发生蓝移（即暴露于溶剂中），观察到的构象变化与Ca2+诱导的相似，这表明在对S-100a蛋白进行光谱研究时，Tb3+可作为Ca2+类似物使用。
