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.

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.

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.