Narasimhan C, Lai C S
Department of Radiology, Medical College of Wisconsin, Milwaukee 53226.
Biopolymers. 1991 Sep;31(10):1159-70. doi: 10.1002/bip.360311004.
We report here a novel approach to label specifically one of the two cryptic, free sulfhydryl groups per subunit of human plasma fibronectin with either an 15N,2H-maleimide spin label or a coumarinylphenyl maleimide fluorescent label. This permits the use of electron spin resonance (ESR) or fluorescence techniques to study molecular dynamics of fibronectin with the label attached to a single site per chain on the protein molecule. The method is based on our observation that upon adsorption of fibronectin to a gelatin-coated surface, the SH1 site, located between the DNA-binding and the cell-binding domains, is partially exposed, while the SH2 site, located within the carboxyl-terminal fibrin-binding domain, remains buried and unreactive. The procedures for the preparation of the selectively labeled fibronectins are described in detail. The physicochemical properties of these single-site labeled fibronectins, particularly as affected by high salt, heparin, surface binding, and temperature, were characterized by ESR spin-label and steady-state fluorescence techniques. The steady-state fluorescence measurement indicates that both local environments of SH1 and SH2 sites are relatively hydrophobic, and that the SH2 site is more hydrophobic than the SH1 site. The ESR results show that heparin or high salt induces an increase in the domainal flexibility in both SH1 and SH2 regions, perhaps through the disruption of domain-domain interactions in the fibronectin molecule, and that the former is more effective than the latter in producing such an effect. The observed heparin effect is reversible by addition of calcium ions in the SH2 regions but not in the SH1 regions. In addition, at temperatures above 44 degrees C, both type III homologous regions containing the free sulfhydryl groups are shown to undergo denaturation and aggregation processes. The data presented here suggest that the newly developed method for differential labeling of the free sulfhydryl groups in fibronectin should be useful for mapping the spatial arrangement of structural domains in the protein molecule using spin-label-spin-probe and fluorescence energy transfer techniques.
我们在此报告一种新方法，可使用15N,2H-马来酰亚胺自旋标记物或香豆素基苯基马来酰亚胺荧光标记物，特异性地标记人血浆纤连蛋白每个亚基的两个隐蔽游离巯基中的一个。这使得能够使用电子自旋共振（ESR）或荧光技术来研究纤连蛋白的分子动力学，其中标记物附着于蛋白质分子每条链上的单个位点。该方法基于我们的观察结果：当纤连蛋白吸附到明胶包被的表面时，位于DNA结合域和细胞结合域之间的SH1位点会部分暴露，而位于羧基末端纤维蛋白结合域内的SH2位点则保持埋藏且无反应性。本文详细描述了制备选择性标记纤连蛋白的步骤。通过ESR自旋标记和稳态荧光技术对这些单位点标记纤连蛋白的物理化学性质进行了表征，特别是受高盐、肝素、表面结合和温度影响的性质。稳态荧光测量表明，SH1和SH2位点的局部环境都相对疏水，且SH2位点比SH1位点更疏水。ESR结果表明，肝素或高盐可能通过破坏纤连蛋白分子中的结构域间相互作用，导致SH1和SH2区域的结构域灵活性增加，且前者在产生这种效应方面比后者更有效。在SH2区域添加钙离子可使观察到的肝素效应逆转，但在SH1区域则不能。此外，在高于44摄氏度的温度下，含有游离巯基的两个III型同源区域均会发生变性和聚集过程。本文提供的数据表明，新开发的纤连蛋白游离巯基差异标记方法，对于使用自旋标记-自旋探针和荧光能量转移技术绘制蛋白质分子中结构域的空间排列可能是有用的。
