Many enzymatic studies aimed at understanding the structure-function-dynamics relationship are conducted under dilute conditions. However, the intracellular environment is highly crowded with biomolecules of varying shapes, sizes, and chemical properties, which can impact a protein’s structure and thereby its function. This discrepancy between scientific study and real-world data can lead to incomplete or misleading conclusions about enzyme behavior in vivo. In the proposed study, we investigate the effects of molecular crowding on Escherichia coli Prolyl-tRNA synthetase (Ec ProRS), a multidomain enzyme responsible for catalyzing the ligation of proline to tRNA Pro during protein biosynthesis. To observe cellular crowding, we employ atomic force microscopy (AFM), a high-resolution scanning probe technique capable of producing nanometer-scale topographic images. AFM enables both qualitative and quantitative analysis of protein samples. Qualitative insights, such as surface roughness and clustering, can reveal structural changes due to crowding, while quantitative measurements of height, area, and volume provide a deeper understanding of protein stability and conformational shifts because of crowding. In this study, we analyze the impact of various crowder molecules, including protein-based crowders (bovine serum albumin and lysozyme) and synthetic polymers, such as polyethylene glycol 20k, on the structure of Ec ProRS. In addition to observing protein crowding, we will present comparative results of AFM studies conducted in air versus in aqueous phase. This approach aims to bridge the gap between conventional dilute-condition studies and the complex, crowded environments in which enzymes naturally operate, offering a more physiologically relevant perspective on enzyme structure and function.
Vlcek et al. (Sun,) studied this question.