Geissinger Süreyya E, Schreiber Andreas, Huber Matthias C, Stühn Lara G, Schiller Stefan M
Zentrum für Biosystemanalyse (ZBSA), University of Freiburg, Habsburgerstraße 49, 79104 Freiburg, Germany.
Faculty of Biology, University of Freiburg, Schänzlestraße 1, 79104 Freiburg, Germany.
ACS Synth Biol. 2020 Apr 17;9(4):827-842. doi: 10.1021/acssynbio.9b00494. Epub 2020 Mar 17.
The investigation of complex biological processes often requires defined multiple bioconjugation and positioning of functional entities on 3D structures. Prominent examples include spatially defined protein complexes in nature, facilitating efficient biocatalysis of multistep reactions. Mimicking natural strategies, synthetic scaffolds should comprise bioorthogonal conjugation reactions and allow for absolute stoichiometric quantification as well as facile scalability through scaffold reproduction. Existing scaffolding strategies often lack covalent conjugations on geometrically confined scaffolds or precise quantitative characterization. Addressing these shortcomings, we present a bioorthogonal dual conjugation platform based on genetically encoded artificial compartments comprising two distinct genetically encoded covalent conjugation reactions and their precise stoichiometric quantification. The SpyTag/SpyCatcher (ST/SC) bioconjugation and the controllable strain-promoted azide-alkyne cycloaddition (SPAAC) were implemented on self-assembled protein membrane-based compartments (PMBCs). The SPAAC reaction yield was quantified to be 23% ± 3% and a ST/SC surface conjugation yield of 82% ± 9% was observed, while verifying the compatibility of both chemical reactions as well as enhanced proteolytic stability. Using tandem mass spectrometry, absolute concentrations of the proteinaceous reactants were calculated to be 0.11 ± 0.05 attomol/cell for PMBC surface-tethered mCherry-ST-His and 0.22 ± 0.09 attomol/cell for PMBC-constituting pAzF-SC-E20F20-His. The established conjugation platform enables quantifiable protein-protein interaction studies on geometrically defined scaffolds and paves the road to investigate effects of scaffold-tethering on enzyme activity.
对复杂生物过程的研究通常需要在三维结构上对功能实体进行明确的多重生物共轭和定位。突出的例子包括自然界中空间定义的蛋白质复合物，其有助于多步反应的高效生物催化。模仿自然策略，合成支架应包含生物正交共轭反应，并允许绝对化学计量定量以及通过支架复制实现简便的可扩展性。现有的支架策略通常缺乏在几何受限支架上的共价共轭或精确的定量表征。为了解决这些缺点，我们提出了一种基于基因编码人工隔室的生物正交双共轭平台，该平台包含两种不同的基因编码共价共轭反应及其精确的化学计量定量。SpyTag/SpyCatcher（ST/SC）生物共轭和可控的应变促进叠氮化物-炔烃环加成（SPAAC）在基于自组装蛋白质膜的隔室（PMBC）上得以实现。SPAAC反应产率经量化为23%±3%，观察到ST/SC表面共轭产率为82%±9%，同时验证了两种化学反应的兼容性以及增强的蛋白水解稳定性。使用串联质谱法，计算得出PMBC表面连接的mCherry-ST-His的蛋白质反应物绝对浓度为0.11±0.05阿托摩尔/细胞，而构成PMBC的pAzF-SC-E20F20-His的绝对浓度为0.22±0.09阿托摩尔/细胞。所建立的共轭平台能够在几何定义的支架上进行可量化的蛋白质-蛋白质相互作用研究，并为研究支架连接对酶活性的影响铺平了道路。
