CONFORMATIONAL PLASTICITY AND ALLOSTERY IN HEMOGLOBIN

变构仍然是分子生物物理学中的一个重要概念，被广泛用于解释许多多亚基蛋白质系统的功能。血红蛋白（Hb）一直是并将继续是典型的变构模型蛋白。虽然Hb反应性的许多特征可以用变构的两态MWC模型来解释，但最近的一些研究表明，这种理想行为存在很大的偏差。解决这些潜在重要偏差的程度和基础的挑战来自分离配体结合中间体的需要和适合测试立体化学模型的位点特异性探针的需要。在这个项目中，几个特定位置的光学探针将用于探测局部的三级构象和全局的四级构象。连续波和时间分辨可见光共振拉曼和近红外吸收将探测血红素及其环境，紫外共振拉曼将探测alpha1beta2界面，一种新的荧光寿命技术将用于探测R-T敏感的2,3- dpg结合位点。光解离COHbs中的双子体重组被用作血红素反应性的探针。这些工具将与新开发的溶胶-凝胶封装技术结合使用，该技术允许捕获非平衡结构。铁金属杂交体和突变体Hbs将与这些技术结合使用，以测试以下五个相互关联的假设：1)两状态模型必须扩展以包含R和T状态族的概念。2)构象可塑性调节变构行为。3) Hb可以作为组合开关，部分由T态内二聚体通信引起。4)提出的通信途径和立体化学模型可以测试从两种状态模型的偏差。5)血红素近端应变的构象调谐调节配体结合。

Allostery remains an important concept in molecular biophysics that is used extensively to explain the functionality in many multi-subunit protein systems. Hemoglobin (Hb) has been and continues to be the prototypical allosteric model protein. Although many features of Hb reactivity are explainable in terms of the two-state MWC model for allostery, several recent studies indicate substantial deviations from this ideal behavior. The challenge in addressing the extent and basis for these potentially important deviations arises from both the need to isolate ligand binding intermediates and the need for site specific probes that are suitable for testing stereochemical models. In this project several site-specific optical probes are to be used to probe both local tertiary conformations and global quaternary states. CW and time-resolved visible resonance Raman and near IR absorption will probe the heme and its environment, UV resonance Raman will probe the alpha1beta2 interface and a new fluorescence lifetime technique will be used to probe the R-T sensitive 2,3-DPG binding site. Geminate recombination in photo-dissociated COHbs is to be used as a probe of heme reactivity. These tools will be used in conjunction with a newly developed sol-gel encapsulation technique which allows for the trapping of non-equilibrium structures. Iron-metal hybrids and mutant Hbs will be used in combination with these techniques to test the following five interrelated hypotheses: 1) The two state model must be expanded to encompass the concept of R and T state families. 2) Conformational plasticity modulates allosteric behavior. 3) Hb can act as a combinatorial switch arising in part from intra-dimer communication within the T state. 4) Proposed communication pathways and stereochemical models that account for deviations from the two state model can be tested. 5) Conformational tuning of proximal strain at the heme modulates ligand binding.