Principles, Methods, and Applications of Protein Folding Inside Cells

The complex mechanism of protein folding in cells has intrigued the scientific community for decades. The physical and chemical forces that drive protein folding have been deciphered by intense experimental, theoretical, and computational methods. Although folding kinetics has been pursued for many proteins in vitro, the crowded cellular environment and the complex solution properties might differentially impact the folding process in vivo. Sampling the native conformation from thousands of folding intermediate states occurs within a timescale of milliseconds to seconds in cells, and replicating this dynamic and highly complex phenomenon under cell-free conditions is an extremely challenging task. The absence of critical regulatory parameters leads to protein misfolding and aggregation. Biophysical approaches like in-cell NMR spectroscopy, in-cell FRET, and FlAsH have facilitated studies focused on analysing protein folding in cells. In this chapter, we discuss the roles of various cellular factors in the protein folding process inside cells, methods to study the folding process in a dynamic cellular environment, and elaborate on the emerging applications of this knowledge to engineer proteins with native-like folds but novel properties of interest.