Molecular Crowding and Enzyme Dynamics: Unraveling the Complex Interactions in Cellular Environments

Abstract

Okinawa Institute of Science and Technology Graduate UniversityDoctor of PhilosophyEnzymes are ubiquitously involved in cellular processes. The enzyme environment conditions greatly affect enzyme specificity, folding and activity. These facts suggest a pivotal role of the cytosolic milieu on enzyme behavior. On the other hand, enzymes could also affect the cytosolic biophysical properties, suggesting an intricated interplay between enzyme catalysis and the cellular milieu. However, this interplay has been poorly studied due to the paramount challenges in mimicking the cellular complexity in vitro and the interdisciplinary nature of these mechanisms. Here, we utilized a liquid-liquid phase separation system to generate membraneless protein droplets, simulating cytosolic protein crowding in vitro. These droplets allow the study of enzyme kinetics in a cytosol-mimicking environment. By using an interdisciplinary approach spanning from biochemistry, rheology and fluid dynamics, we characterized the interplay between enzyme activity and a cytosolic-mimicking droplets. Specifically, we showed how enzymatic activity is affected by the cytosolic features and environmental stress in protein-crowded conditions. Moreover, we discovered that enzyme can generate emerging phenomena when localized in a protein-crowded environment such as droplets migration and viscosity modulation. This study highlights the interconnection between enzymes and the cytosolic environment and the underestimated role of enzyme activity in triggering complex cellular phenomena.doctoral thesi