Protein Language Models (pLMs) generate per-protein embeddings that encode functional, structural, and evolutionary information, yet the relationships captured in these representations remain difficult to explore systematically. ProtSpace (https://protspace.app) is a web application for interactive visualization of pLM embedding spaces, enabling hypothesis generation directly in the browser without installation. Unlike traditional network-based tools that exclusively visualize amino acid sequence similarity, ProtSpace explores embedding spaces, revealing relationships often not captured by traditional comparisons. Users provide protein sequences or pre-computed embeddings through a Google Colab notebook or the Python CLI; the pipeline applies dimensionality reduction, retrieves 38 annotation types spanning UniProt, InterPro, NCBI Taxonomy, TED structural domains, and sequence-based predictors served via Biocentral, and produces a portable binary file for the browser-based viewer. WebGL-accelerated rendering supports interactive exploration of over 570,000 proteins. Distinctive features include per-point pie charts for multi-label annotations and integrated 3D structure viewing through AlphaFold2 predictions. All computation happens on the user's machine, ensuring data privacy. We demonstrate the utility of ProtSpace through a progressive zoom-in across biological scales: from global proteome organization of Swiss-Prot, through cross-species comparison revealing conserved and lineage-specific families, to functional hypothesis generation within the beta-lactamase superfamily. ProtSpace is freely available at https://protspace.app under the Apache 2.0 license.

The continuous discovery and engineering of polyethylene terephthalate (PET) hydrolases are critical to advancing sustainable plastic recycling. A significant number of PET hydrolases have been identified to date; nonetheless, high-throughput screening and evaluation of enzyme characteristics remain a key bottleneck in protein engineering. This study develops an ultra-high-throughput fluorescence-activated droplet sorting (FADS) system for screening PET hydrolases, based on pH sensing. The pH change caused by the released depolymerization product, terephthalic acid (TPA), is correlated with the fluorescent variation of the pH-sensitive C-SNARF-4F probe. We applied this method to screen mutant libraries of two PET hydrolases, DepoPETase β and a new enzyme, SdPETase (derived from Saccharopolyspora dendranthemae), identified via genome mining. Variants exhibiting 1.21-fold and 2.65-fold higher hydrolytic activities were successfully obtained for DepoPETase β and SdPETase, respectively. The successful integration of the pH-based assay with FADS highlights its versatility and efficiency for ultra-high-throughput screening of PET hydrolases.

Adenosine triphosphate (ATP) serves as the universal energy currency in cellular metabolism. However, real-time analysis of ATP dynamics in prokaryotes remains a challenge due to significant intracellular pH fluctuations and high background interference. To address this, we developed IGAS, a novel genetically encoded biosensor engineered by integrating a binding protein derived from Bacillus subtilis PS3 with the acid-resistant fluorescent protein cpmCherry and miRFP670nano3. Characterization revealed that IGAS exhibits a robust 2.8-fold dynamic range, high selectivity for ATP, and remarkable pH stability. When expressed in E. coli, IGAS enabled real-time monitoring of intracellular ATP fluctuations throughout the bacterial growth cycle, demonstrating high consistency with standard luciferase assays. Furthermore, guided by molecular dynamics (MD) simulations, we identified key residues to engineer IGAS variants with tunable affinities. These sensors were successfully applied to diverse cellular environments, ranging from cytoplasmic targeting to mammalian cell surface display. Collectively, our results demonstrate the excellent reversibility and versatility of IGAS, establishing it as a powerful tool for dynamic ATP detection in complex biological systems.

Synthetic cells, assembled from defined molecular components, are designed to mimic the features, form, and function of living cells. Light has emerged as a uniquely precise, biorthogonal, and non-invasive stimulus for regulating and energizing these systems, enabling chemical inhomogeneity and an out-of-equilibrium state central to many cellular processes. This review highlights the biological behaviors and functions that light has helped recreate in synthetic cells, including compartmentalization, energy supply and metabolism, protein synthesis, communication, growth, shape change and division, and motility. We survey the breadth of light-responsive components incorporated into synthetic cells, spanning photoswitchable and photocleavable small molecules, photoswitchable proteins, photocatalysts, nanoparticles, and photosynthetic organelles or organisms. Finally, we offer a perspective on key design considerations such as wavelength, reversibility, integration, biocompatibility, multicolor regulation, and biohybrid strategies. Together, these advances chart promising routes toward more dynamic, energy-autonomous, and programmable synthetic cells that will deepen our understanding of cellular functions and enable emerging biotechnological applications.