NMR and DFT Investigation of Porphyrin Conformation in Cytochromes c

描述（由申请人提供）：该项目的长期目标是开发第一个能够定量测量溶液中血红素构象的实验工具。该工具将对NIH路线图的结构生物学倡议做出重要贡献，可用于未来确定血红素蛋白核磁共振（NMR）溶液结构的研究。血红素蛋白具有多种生物功能，包括：电子传输、气体传输、小分子传感和催化。每个血红素蛋白优化血红素辅助因子的反应性，以实现其特定的功能和血红素构象可以说是最未被研究的策略。了解这一决定血红素电子结构和反应性的因素对于全面理解涉及血红素蛋白的生化过程以及与血红素蛋白功能障碍相关的疾病至关重要。细胞色素c蛋白家族将被用作开发该工具的模型系统，因为该家族利用非平面血红素辅助因子和蛋白质基质提供了一种策略，以系统的方式改变血红素构象，不太可能引入大规模的蛋白质构象变化。顺磁1H和13C核磁共振波谱将被用作光谱探针，因为这种技术可以同时检测卟啉大环上血红素的几个位置的电子结构。多维核磁共振实验和血红素辅助因子的选择性同位素富集将用于血红素顺磁1H和13C共振分配。密度泛函理论（DFT）计算，最初由x射线晶体学和共振拉曼光谱辅助，将用于根据血红素辅因子的面外扭曲来解释顺磁核磁共振数据。最终，该策略将在顺磁核磁共振数据和血红素构象之间产生基于dft的相关性。这种新工具可以应用于生物系统中具有重要功能的血红素蛋白的广泛范围。基于dft的相关性还将识别血红素构象、电子结构和反应性之间的关系。公共卫生相关性：包括人类在内的生物有机体使用血红素进行一系列不同的基本过程。为了使特定过程的效率最大化，生物系统操纵血红素及其周围环境。可以说，对血红素蛋白最未被充分研究的操作是改变其形状，而这一提议将开发出第一种测量溶液中血红素形状的方法。

DESCRIPTION (provided by applicant): The long-term objective of this project is to develop the first experimental tool capable of quantitatively measuring the conformation of heme in solution. This tool will be an important contribution to the structural biology initiative of the NIH roadmap, which could be used in future studies that determine the nuclear magnetic resonance (NMR) solution structure of heme proteins. Heme proteins have a diverse set of biological functions, including: electron transport, gas transport, small molecule sensing, and catalysis. Each heme protein optimizes the reactivity of the heme cofactor to achieve its specific function and heme conformation is arguably the most under-investigated strategy available. It is essential to understand this determinant of heme electronic structure and reactivity to fully comprehend the biochemical processes involving heme proteins, and diseases associated with their malfunction. The cytochrome c family of proteins will be used as a model system to develop this tool because this family utilizes a nonplanar heme cofactor and the protein matrix provides a strategy to alter the heme conformation in a systematic way that is unlikely to introduce large-scale protein conformational changes. Paramagnetic 1H and 13C NMR spectroscopy will be used as a spectroscopic probe because this technique can simultaneously examine the electronic structure of heme at several positions on the porphyrin macrocycle. Multi-dimensional NMR experiments and selective isotopic enrichment of the heme cofactor will be used to make heme paramagnetic 1H and 13C resonance assignments. Density functional theory (DFT) calculations, initially assisted by X-ray crystallography and resonance Raman spectroscopy, will be used to interpret the paramagnetic NMR data in terms of out-of-plane distortions of the heme cofactor. Ultimately, this strategy will produce a DFT-based correlation between paramagnetic NMR data and heme conformation. This new tool can be applied to a wide range of heme proteins that have important functions within biological systems. The DFT-based correlation will also identify the relationship between heme conformation, electronic structure, and reactivity. PUBLIC HEALTH RELEVANCE: Biological organisms, including humans, use heme to carry out a diverse set of fundamental processes. To maximize the efficiency of a specific process, biological systems manipulate heme and its surroundings. Arguably, the most under-investigated manipulation available to heme proteins is changing the shape, and this proposal will develop the first method to measure the shape of heme in solution.