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Precision design of single and multi-heme de novo proteins
bioRxiv - Synthetic Biology Pub Date : 2020-09-24 , DOI: 10.1101/2020.09.24.311514
George H. Hutchins , Claire E. M. Noble , Hector Blackburn , Ben Hardy , Charles Landau , Alice E. Parnell , Sathish Yadav , Christopher Williams , Paul R. Race , A. Sofia F. Oliveira , Matthew P. Crump , Christiane Berger-Schaffitzel , Adrian J. Mulholland , J. L. Ross Anderson

The de novo design of simplified porphyrin-binding helical bundles is a versatile approach for the construction of valuable biomolecular tools to both understand and enhance protein functions such as electron transfer, oxygen binding and catalysis. However, the methods utilised to design such proteins by packing hydrophobic side chains into a buried binding pocket for ligands such as heme have typically created highly flexible, molten globule-like structures, which are not amenable to structural determination, hindering precise engineering of subsequent designs. Here we report the crystal structure of a de novo two-heme binding maquette protein, 4D2, derived from the previously designed D2 peptide, offering new opportunities for computational design and re-engineering. The 4D2 structure was used as a basis to create a range of heme binding proteins which retain the architecture and stability of the initial crystal structure. A well-structured single-heme binding variant was constructed by computational sequence redesign of the hydrophobic protein core, assessed by NMR, and utilised for experimental validation of computational redox prediction and design. The structure was also extended into a four-heme binding helical bundle resembling a molecular wire. Despite a molecular weight of only 24kDa, imaging by CryoEM illustrated a remarkable level of detail in this structure, indicating the positioning of both the secondary structure and the heme cofactors. The design and determination of atomic-level resolution in such de novo proteins is an invaluable resource for the continued development of novel and functional protein tools.

中文翻译:

单和多血红素从头蛋白质的精确设计

简化的卟啉结合螺旋束的从头设计是一种通用的方法,可用于构建有价值的生物分子工具,以了解和增强蛋白质功能,例如电子转移,氧结合和催化作用。然而,用于通过将疏水性侧链包装到诸如配体诸如血红素的埋藏的结合袋中来设计此类蛋白质的方法通常产生了高度柔性的,熔融的球状结构,其不适于结构确定,这妨碍了后续设计的精确工程设计。 。在这里,我们报道了从先前设计的D2肽衍生而来的从头开始的两血红素结合模样蛋白4D2的晶体结构,为计算设计和再工程提供了新的机会。4D2结构被用作创建一系列血红素结合蛋白的基础,这些蛋白保留了初始晶体结构的结构和稳定性。通过疏水蛋白核心的计算序列重新设计,构建结构良好的单血红素结合变异体,通过NMR进行评估,并用于计算氧化还原预测和设计的实验验证。该结构还扩展为类似于分子丝的四血红素结合螺旋束。尽管分子量仅为24kDa,但CryoEM成像显示该结构的细节水平非常高,表明二级结构和血红素辅因子的定位。
更新日期:2020-09-25
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