Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.

中文翻译：

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碳酸酐酶II活性的光学控制的预测方法。

光遗传学和光药理学是研究生化系统的有力方法。前者基于利用大量发色团的遗传编码感光体，而后者则依赖于可自由扩散或共价连接至通常为天然或工程半胱氨酸的特定生物缀合位点的合成光电开关。合适的半胱氨酸位点和合适的连接接头的鉴定通常是一个漫长而繁琐的过程。在这里，我们描述了一个计算机筛选方法，旨在提出少量的最佳组合。通过将这种计算方法应用于人碳酸酐酶和一组三个光致变色的束缚配体，潜在位点-配体组合的数量从750多种减少到6种，然后我们进行了实验评估。这六个组合中的两个组合产生了光响应性人碳酸酐酶（LihCAs），其通过酶活性测定，质谱和X射线晶体学进行了表征。我们的研究还提供了对半胱氨酸对马来酰亚胺的反应性和获得的加合物的水解稳定性的见解。