Research Projects

The trigger factor (TF) is the only ribosome-associated chaperone known in bacteria, as it is the first to interact with the nascent polypeptide chain and does not need ATP for its function. It has three domains with specific functions. Deletion of the TF gene leads to severe protein aggregation inside cells, highlighting its importance. Cellular crowding adds complexity, leading to structural alterations, changes in function and dynamics, and enhanced protein aggregation. Understanding how the trigger factor is affected by the crowded milieu is crucial for its proper functioning. TF, a 48 kDa protein, has rich dynamics necessary for its chaperone action. However, there is limited information on its structure-function-dynamics correlation in the crowded scenario. This proposal aims to conduct an in-depth biophysical study of the TF chaperone, both in free and ribosome-bound form, in presence of macromolecular crowding agents, both synthetic and protein-based. The approach includes probing the conformational distribution of TF at both the ensemble and single molecule level using FRET, folding and unfolding kinetics using the stopped-flow technique, diffusion and fast dynamics using fluorescence correlation spectroscopy (FCS), and extensive binding studies with unfolded proteins and the ribosome. The findings aim to provide a comprehensive overview of the structural and functional landscape of the chaperone when in isolation and during its interaction with unfolded/misfolded proteins and the ribosomal unit. Further studies should be conducted in cell mimicking environments with different crowders and their intrinsic molecular properties to gain a deeper understanding of the trigger factor in bacteria.