Zinah Hilal Khaleel

The design of organic–peptide hybrids has the potential to combine our vast knowledge of protein design with small molecule engineering to create hybrid structures with complex functions. Here, we describe the computational design of a photoswitchable Ca ²⁺ -binding organic–peptide hybrid. The designed molecule, designated Ca ²⁺ -binding switch (CaBS), combines an EF-hand motif from classical Ca ²⁺ -binding proteins such as calmodulin with a photoswitchable group that can be reversibly isomerized between a spiropyran (SP) and merocyanine (MC) state in response to different wavelengths of light. The MC/SP group acts both as a photoswitch as well as an optical sensor of Ca ²⁺ binding. Photoconversion of the SP to the corresponding MC unmasks an acidic phenol, which CaBS uses as an integral part of both its Ca ²⁺ -binding site as well as its tertiary and quaternary structure. By design, the SP state of CaBS is monomeric, while the Ca ²⁺ -bound form of the MC state is an obligate dimer, with two Ca ²⁺ -binding sites formed at the interface of a domain-swapped dimer. Thus, light and Ca ²⁺ were expected to serve as an “AND gate” that powers a change in backbone structure/dynamics, oligomerization state, and fluorescence properties of the designed molecule. CaBS was designed using Rosetta and molecular dynamics simulations, and experimentally characterized by nuclear magnetic resonance, isothermal titration calorimetry, and optical titrations. These data illustrate the potential of combining small molecule engineering with de novo protein design to develop sensors whose conformation, association state, and optical properties respond to multiple environmental cues.

Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure‐guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR‐associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo‐electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure‐guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.