CONFORMATIONAL PLASTICITY AND ALLOSTERY IN HEMOGLOBIN

血红蛋白的构象可塑性和变构

• 批准号: 6410452
• 负责人: JOEL M FRIEDMAN
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410452
• 关键词: Raman spectrometry; allosteric site; chemical models; conformation; hemoglobin; hemoprotein structure; infrared spectrometry; laser spectrometry; protein isoforms; protein structure function

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.

变构仍然是分子生物物理学中的一个重要概念，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。尽管 Hb 反应性的许多特征可以用变构的两态 MWC 模型来解释，但最近的几项研究表明与这种理想行为存在很大偏差。解决这些潜在重要偏差的程度和基础的挑战源于分离配体结合中间体的需要以及适合测试立体化学模型的位点特异性探针的需要。在该项目中，将使用多个位点特定的光学探针来探测局部三级构象和全局四级态。 CW 和时间分辨可见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，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。光解离的 COHb 中的双子重组可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，该技术可以捕获非平衡结构。铁金属杂化物和突变型 Hb 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）必须扩展两种状态模型以包含 R 和 T 状态族的概念。 2）构象可塑性调节变构行为。 3) Hb 可以充当组合开关，部分源于 T 状态内二聚体内的通信。 4）可以测试所提出的解释两种状态模型偏差的通信路径和立体化学模型。 5) 血红素近端应变的构象调整调节配体结合。