The WW-domain scaffold as a model system for the de novo design of miniaturized phosphate receptors, phosphatases and sulfatases

WW 结构域支架作为微型磷酸盐受体、磷酸酶和硫酸酯酶从头设计的模型系统

• 批准号: 414261058
• 负责人: Professorin Dr. Franziska Thomas
• 金额: --
• 依托单位: Organisch-Chemisches Institut
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/414261058?language=en
• 关键词: WW; domain; scaffold; model; system

Enzymes catalyze most of the biochemical processes in living systems, and this with astonishing substrate specificity, stereoselectivity and efficiency. Hence, it is no surprise that efforts are made to use enzymes for chemical synthesis. Next of using native enzymes for chemical reactions, engineering of catalytic properties on protein scaffolds or designing enzymes completely from scratch is of increasing scientific interest. Most strategies in enzyme design comprise redesign of natural protein scaffolds. The advantage resides in the huge number of available protein structures. However, the sequence-to-structure relationships of most protein scaffolds are not yet understood, and modifications might lead to protein misfolding. In small, well-characterized protein folding motifs the sequence positions, which allow modification, are known and these structures are accessible by chemical synthesis. On the downside, few protein folds are thoroughly studied and only the coiled-coil motif is well-understood. Consequently, mostly α-helical bundles were previously used for the de novo design of miniaturized enzymes.This research concept is centered on the design of miniaturized enzymes. The WW domain, a small, three-stranded β-sheet protein folding motif, will be investigated as potential alternative scaffold in the design of mini enzymes. In preliminary studies, sequence alignments and alanine scans of the ligand binding site were used to identify amino acid residues, which are relevant for structure and/or function. Based on this, designed sequences are proposed, which encode for a basic WW-domain scaffold. This scaffold is the starting point for the design of WW domains with different binding properties or even catalytic activity. As a proof-of-concept, the de novo design of representatives of the main WW-domain groups is planned. Furthermore, WW domains showing binding to phosphorylated peptides and phosphorylated organic molecules will be created. This in turn is a starting point for the de novo design of WW-domain based phosphatases and sulfatases. To identify active WW-domain scaffolds, a combinatorial approach based on the reconstitution of split WW domains by coiled-coil association is anticipated. For this purpose, the WW-domain fragments are linked to the strands of an antiparallel coiled coil, respectively. Previously, this concept was successfully tested with the split WW domain of PIN1.The presented research project is understood as initiator for the design of further ß-sheet-based protein folding motifs with catalytic activity. In future studies catalysis of ligation reactions, phophorylations and transfer reactions will be of interest.

酶催化生物系统中的大多数生化过程，并具有惊人的底物特异性，立体选择性和效率。因此，努力将酶用于化学合成也就不足为奇了。下一个使用天然酶进行化学反应，催化性能在蛋白质支架上的工程或完全从头开始设计酶是越来越多的科学意义。酶设计中的大多数策略完全重新设计了天然蛋白质支架。该优势属于大量可用蛋白质结构。但是，大多数蛋白质支架的序列与结构关系尚未了解，并且修饰可能导致蛋白质错误折叠。在小的，良好的蛋白质折叠基序中，允许修饰的序列位置已知，并且可以通过化学合成访问这些结构。不利的一面是，很少有蛋白质折叠是彻底研究的，只有盘绕螺旋的图案才理解。因此，以前主要用于微型酶的从头设计。该研究概念以微型酶的设计为中心。 WW结构域是一种小的三链β-折叠蛋白折叠基序，将被研究为迷你酶设计中的潜在替代支架。在初步研究中，配体结合位点的序列比对和丙氨酸扫描用于鉴定与结构和/或功能相关的氨基酸残基。基于此，提出了设计的序列，该序列编码为基本的WW域支架。该支架是具有不同结合特性甚至催化活性的WW域设计的起点。作为概念验证，计划了代表主要WW域组的从头设计。此外，将创建与磷酸化的胡椒体和磷酸化有机分子结合的WW结构域。反过来，这是基于WW域的磷酸酶和硫酸酶的从头设计的起点。为了识别活跃的WW域支架，预计将通过盘绕圈结合结合基于拆分WW域的重建的组合方法。为此，WW域片段分别与反平行线圈线圈的链有关。以前，该概念已通过PIN1的分裂WW域成功测试。提出的研究项目被理解为设计具有催化活性的进一步基于β-折叠的蛋白质折叠基序的发起者。在未来的研究中，连接反应的催化作用将引起人们的关注。