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

酶催化生命系统中的大多数生化过程，并且具有惊人的底物特异性、立体选择性和效率。因此，人们努力利用酶进行化学合成也就不足为奇了。其次，利用天然酶进行化学反应，蛋白质支架上的催化性能工程或完全从零开始设计酶正在引起越来越多的科学兴趣。酶设计中的大多数策略包括对天然蛋白质支架的重新设计。其优势在于大量可用的蛋白质结构。然而，大多数蛋白质支架的序列-结构关系尚不清楚，修饰可能导致蛋白质错误折叠。在小的、特征明确的蛋白质折叠基序中，允许修饰的序列位置是已知的，这些结构可以通过化学合成获得。缺点是，很少有蛋白质折叠被彻底研究，只有卷曲的线圈基序被很好地理解。因此，大多数α-螺旋束先前被用于小型化酶的从头设计。这一研究理念的核心是小型化酶的设计。WW结构域是一个小的，三链β-片蛋白折叠基序，将被研究作为设计迷你酶的潜在替代支架。在初步研究中，利用配体结合位点的序列比对和丙氨酸扫描来鉴定与结构和/或功能相关的氨基酸残基。在此基础上，提出了编码基本ww结构域支架的设计序列。该支架是设计具有不同结合特性甚至催化活性的WW结构域的起点。作为概念验证，规划了主要www域组代表的从头设计。此外，WW结构域将与磷酸化肽和磷酸化有机分子结合。这反过来又是基于www结构域的磷酸酶和硫酸盐酶的重新设计的起点。为了识别有活性的WW结构域支架，我们期望一种基于螺旋-螺旋关联对分裂的WW结构域进行重组的组合方法。为此，w -域片段分别连接到反平行线圈的链上。之前，这个概念已经在PIN1的分割WW域上成功测试过了。提出的研究项目被理解为进一步设计具有催化活性的ß-sheet-based蛋白质折叠基序的启动器。在未来的研究中，将对连接反应、磷酸化和转移反应的催化作用感兴趣。

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.