Young Hyun No

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.

The adsorption of peptides and proteins on hydrophobic solid surfaces has received considerable research attention owing to their wide applications to biocompatible nanomaterials and nanodevices, such as biosensors and cell adhesion materials with reduced nanomaterial toxicity. However, fundamental understandings about physicochemical hydrophobic interactions between peptides and hydrophobic solid surfaces are still unknown. In this study, we investigate the effect of secondary structures on adsorption energies between peptides and hydro-phobic solid surfaces via experimental and theoretical analyses using surface-assisted laser desorption/ionization-time-of-flight (SALDI-TOF) and molecular dynamics (MD) simulations. The hydrophobic interactions between peptides and hydrophobic solid surfaces measured via SALDI-TOF and MD simulations indicate that the hydrophobic interaction of peptides with random coil structures increased more than that of peptides with an alpha-helix structure when polar amino acids are replaced with hydrophobic amino acids. Additionally, our study sheds new light on the fundamental understanding of the hydrophobic interaction between hydrophobic solid surfaces and peptides that have diverse secondary structures.

Peptide assemblies have received significant attention because of their important role in biology and applications in bionanotechnology. Despite recent efforts to elucidate the principles of peptide self-assembly for developing novel functional devices, peptide self-assembly on two-dimensional nanomaterials has remained challenging. Here, we report nature-inspired two-dimensional peptide self-assembly on pristine graphene via optimization of peptide–peptide and peptide–graphene interactions. Two-dimensional peptide self-assembly was designed based on statistical analyses of >10⁴ protein structures existing in nature and atomistic simulation-based structure predictions. We characterized the structures and surface properties of the self-assembled peptide formed on pristine graphene. Our study provides insights into the formation of peptide assemblies coupled with two-dimensional nanomaterials for further development of nanobiocomposite devices.