Veenstra T D, Johnson K L, Tomlinson A J, Craig T A, Kumar R, Naylor S
Department of Nephrology Research Unit, Mayo Clinic Foundation, Rochester, Minnesota, USA.
J Am Soc Mass Spectrom. 1998 Jan;9(1):8-14. doi: 10.1016/S1044-0305(97)00229-8.
Electrospray ionization mass spectrometry (ESI-MS) was used to measure conformational changes within the DNA-binding domain of the vitamin D receptor (VDR DBD) upon binding zinc (Zn2+). As increasing concentrations of Zn2+ were added to the VDR DBD, a gradual shift in the mass envelope to lower charge states was observed in the multiply charged spectrum. The shift in the charge states was correlated to changes observed in the far-ultraviolet circular dichroic (far-UV CD) spectrum of the protein as it was titrated with Zn2+. Both the multiply charged ESI and far-UV CD spectra of the Zn(2+)-titrated protein show that the binding of the first Zn2+ ion to the protein results in very little conformational change in the protein. The binding of a second Zn2+ ion resulted in a significant alteration in the structure of the protein as indicated by changes in both the multiply charged ESI and far-UV CD spectra. Much smaller changes were seen within the multiply charged ESI or far-UV CD spectra upon increasing the Zn2+ concentration beyond 2 mol/mol of protein. The results presented indicate that ESI-MS in combination with CD is a powerful method to measure gross conformational changes induced by the binding of metals to metalloproteins.
采用电喷雾电离质谱（ESI-MS）来测定维生素D受体（VDR DBD）的DNA结合结构域在结合锌（Zn2+）时的构象变化。随着向VDR DBD中添加的Zn2+浓度增加，在多电荷谱中观察到质量包络逐渐向较低电荷态移动。电荷态的变化与在用Zn2+滴定蛋白质时其远紫外圆二色性（远紫外CD）光谱中观察到的变化相关。Zn(2+)滴定蛋白质的多电荷ESI光谱和远紫外CD光谱均表明，第一个Zn2+离子与蛋白质的结合导致蛋白质构象变化很小。第二个Zn2+离子的结合导致蛋白质结构发生显著改变，这在多电荷ESI光谱和远紫外CD光谱的变化中均有体现。当Zn2+浓度增加到超过蛋白质的2 mol/mol时，在多电荷ESI光谱或远紫外CD光谱中观察到的变化要小得多。所呈现的结果表明，ESI-MS与CD相结合是一种测量金属与金属蛋白结合所诱导的总体构象变化的有力方法。
