Keown M B, Henry A J, Ghirlando R, Sutton B J, Gould H J
The Randall Institute, King's College London, UK.
Biochemistry. 1998 Jun 23;37(25):8863-9. doi: 10.1021/bi972354h.
We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.
我们已采用等温滴定量热法（ITC）和圆二色性（CD）光谱法来表征免疫球蛋白E（IgE）的可溶性片段（IgE-Fc和Fcε3-4）与高亲和力受体FcεRIα链的可溶性片段（sFcεRIα）之间的结合。使用ITC测定的IgE-Fc和Fcε3-4与sFcεRIα相互作用的热力学参数，证实了先前的结论，即Cε2结构域不参与相互作用，且两种复合物的化学计量比均为1:1。对于IgE-Fc和Fcε3-4，在37.3℃时，ΔH°值为-36.9±4.6 kcal mol-1，ΔCp°值为-820±120 cal mol-1 K-1。ΔS°为零的温度是284±1 K，这表明在生理温度下，熵对缔合热力学的贡献是不利的。特别值得关注的是ΔCp°的较大值。先前对这两种蛋白质进行的诱变研究表明，参与复合物形成的IgE和FcεRIα的表面积较大，这可能部分解释了ΔCp°的大小。其他贡献可能来自结合位点内的水合作用以及复合物各个组分三级结构的变化。然而，IgE、IgE-Fc和Fcε3-4与sFcεRIα形成的复合物的CD光谱仅仅是其各个组分光谱的总和，这表明免疫球蛋白结构域折叠的二级结构在复合物形成时得以保留。因此，三级结构的任何变化都必须局限于IgE中免疫球蛋白结构域Cε3和Cε4以及FcεRIα链中两个免疫球蛋白样结构域的相对位置。
