Molecular handcraft of a well-folded protein chimera.

Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous 'copy-paste' processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/ß-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.

Visualizing Molecular Architectures of Cellular Condensates: Hints of Complex Coacervation Scenarios.

In the last decade, liquid-liquid phase separation has emerged as a fundamental principle in the organization of crowded cellular environments into functionally distinct membraneless compartments. It is now established that biomolecules can condense into various physical phases, traditionally defined for simple polymer systems, and more recently elucidated by techniques employed in life sciences. We review pioneering cryo-electron tomography studies that have begun to unravel a wide spectrum of molecular architectures, ranging from amorphous to crystalline assemblies, that underlie cellular condensates. These observations bring into question current interpretations of microscopic phase behavior. Furthermore, by examining emerging concepts of non-classical phase separation pathways in small-molecule crystallization, we draw parallels with biomolecular condensation that highlight aspects not yet fully explored. In particular, transient and metastable intermediates that might be challenging to capture experimentally inside cells could be probed through computational simulations and enable a multi-scale understanding of the subcellular organization governed by distinct phases.

Mutational analysis of protein folding inside the ribosome exit tunnel.

Recent work has demonstrated that cotranslational folding of proteins or protein domains in, or in the immediate vicinity of, the ribosome exit tunnel generates a pulling force on the nascent polypeptide chain that can be detected using a so-called translational arrest peptide (AP) engineered into the nascent chain as a force sensor. Here, we show that AP-based force measurements combined with systematic Ala and Trp scans of a zinc-finger domain that folds in the exit tunnel can be used to identify the residues that are critical for intraribosomal folding. Our results suggest a general approach to characterize the folded state(s) that may form as a protein domain moves progressively down the ribosome exit tunnel.