Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues.
Designer Heme Proteins: Achieving Novel Function with Abiological Heme Analogues.
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DOI：
10.1021/acs.accounts.1c00588
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发表时间：
2021-12-21
影响因子：
18.3
通讯作者：
Marletta MA
中科院分区：
文献类型：
--
作者：
Lemon CM;Marletta MA
血红素蛋白已被证明是开发具有新功能的设计蛋白的便捷平台。这是通过用具有所需性质的血红素类似物取代天然的铁卟啉辅因子来实现的。用另一种金属取代卟啉的铁中心，为实现新的蛋白质功能提供了一条途径。一种较少探索的方法是用替代的四吡咯大环或相关的配体取代卟啉辅因子。一般来说，这些配体表现出不同于卟啉的化学性质和反应活性。虽然这些技术主要用于开发人造金属酶，但这种方法在生物化学、健康和医学问题上也有许多其他应用。在蛋白质环境中加入合成的辅因子是一种简单的方式来提高水溶性和生物相容性。它绕过了费力合成水溶性辅因子的过程，后者往往会引入大量电荷，导致不受欢迎的生物积累。为此，在血红素蛋白中加入非天然辅因子使设计的蛋白质得以开发，如光学氧传感器、核磁共振造影剂、光谱探测器、询问蛋白质功能的工具、抗生素和荧光蛋白质。人工辅因子的掺入通常是通过去除血红素使全蛋白变性来完成的；然后，重折叠的载脂蛋白与人工辅因子重组。这一过程通常会导致大量的蛋白质丢失，并不一定保证复性的蛋白质采用天然结构。为了绕过这些问题，我们的实验室率先使用了大肠杆菌RP523菌株，使用基于表达的方法将人工辅助因子整合到血红素蛋白中。该菌株缺乏生物合成血红素的能力，细菌细胞壁对血红素和相关分子具有渗透性。通过这种方式，在生长介质中补充的血红素类似物被结合到血红素蛋白中。这种方法也可以用于直接表达脱辅基蛋白，以用于随后的重组。这些方法已被用来将非天然辅助因子整合到能抵抗恶劣环境条件的血红素蛋白中：来自CaldanAerbacter subterraneus(Cs)的血红素一氧化氮/氧结合蛋白(H-NOx)和来自铜绿假单胞菌(PA)的血红素获取系统蛋白A(HasA)。这些蛋白质的特殊稳定性使它们成为生物医学应用的理想支架。光学氧传感已经完成，使用磷光体，作为人工血红素辅因子的钌卟啉。顺磁性锰和格拉卟啉可产生高弛豫度、基于蛋白质的磁共振造影剂。荧光磷科罗尔用作血红素类似物来产生荧光蛋白。与HasA结合的非卟啉辅因子铁络合物可抑制病原菌的生长。此外，HasA可以向细菌胞浆中传递一种镓酞菁，作为光化学杀菌的敏化剂。总而言之，这些例子说明了设计血红素蛋白的潜力，以解决健康和医学领域中迅速增长的问题。本文提出的概念和方法可以扩展到下一代生物医学传感和成像试剂的开发，以识别和量化临床上相关的代谢物和其他关键疾病生物标记物。
Heme proteins have proven to be a convenient platform for the development of designer proteins with novel functionalities. This is achieved by substituting the native iron porphyrin cofactor with a heme analog that possesses the desired properties. Replacing the iron center of the porphyrin with another metal provides one inroad to novel protein function. A less explored approach is substitution of the porphyrin cofactor with an alternative tetrapyrrole macrocycle or a related ligand. In general, these ligands exhibit chemical properties and reactivity that are distinct from porphyrins. While these techniques have most prominently been utilized to develop artificial metalloenzymes, there are many other applications of this methodology to problems in biochemistry, health, and medicine. Incorporation of synthetic cofactors into protein environments represents a facile way to impart water solubility and biocompatibility. It circumvents the laborious synthesis of water-soluble cofactors, which often introduces substantial charge that leads to undesired bioaccumulation. To this end, the incorporation of unnatural cofactors in heme proteins has enabled the development of designer proteins as optical oxygen sensors, MRI contrast agents, spectroscopic probes, tools to interrogate protein function, antibiotics, and fluorescent proteins. Incorporation of an artificial cofactor is frequently accomplished by denaturing the holoprotein with removal of the heme; the refolded apoprotein is then reconstituted with the artificial cofactor. This process often results in substantial protein loss and does not necessarily guarantee that the refolded protein adopts the native structure. To circumvent these issues, our laboratory has pioneered the use of the RP523 strain of E. coli to incorporate artificial cofactors into heme proteins using expression-based methods. This strain lacks the ability to biosynthesize heme, and the bacterial cell wall is permeable to heme and related molecules. In this way, heme analogs supplemented in the growth media are incorporated into heme proteins. This approach can also be leveraged for the direct expression of the apoprotein for subsequent reconstitution. These methodologies have been exploited to incorporate non-native cofactors into heme proteins that are resistant to harsh environmental conditions: the heme nitric oxide/oxygen binding protein (H-NOX) from Caldanaerobacter subterraneus (Cs) and the heme acquisition system protein A (HasA) from Pseudomonas aeruginosa (Pa). The exceptional stability of these proteins makes them ideal scaffolds for biomedical applications. Optical oxygen sensing has been accomplished using a phosphorescent ruthenium porphyrin as the artificial heme cofactor. Paramagnetic manganese and gadolinium porphyrins yield high-relaxivity, protein-based MRI contrast agents. A fluorescent phosphorus corrole serves as a heme analog to produce fluorescent proteins. Iron complexes of non-porphyrin cofactors bound to HasA inhibit the growth of pathogenic bacteria. Moreover, HasA can deliver a gallium phthalocyanine into the bacterial cytosol to serve as a sensitizer for photochemical sterilization. Together, these examples illustrate the potential for designer heme proteins to address burgeoning problems in the areas of health and medicine. The concepts and methodologies presented in this Account can be extended to the development of next-generation biomedical sensing and imaging agents to identify and quantify clinically relevant metabolites and other key disease biomarkers.
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影响因子：
18.3
作者：
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通讯作者：
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