Biophysical Aspects of Co- and Post-Translational Protein Folding

Proteins perform biological functions that are crucial to enable, support and protect life. Protein shape is extraordinarily important because it determines biological activity. The goal of this project is to reveal how proteins achieve their complex and highly organized three-dimensional shape in the context of living cells.

This process is commonly referred to as protein folding. This is an intricate process, and it occurs at the level of individual molecules in the complex cellular environment. Unfortunately, very little is still known about how proteins fold in the biological milieu.

As part of this project, the high-resolution structure of the ribosome, the protein-making machinery, as it binds and generates proteins will be mapped. With the help of sophisticated lasers, magnets and electron beams, how the nascent protein interacts with the ribosome and how the ribosome in turn helps the protein fold is expected to be revealed.

This process is similar to picture-taking at the molecular level. The process of protein making in real time and watching 3D-protein-shape formation as it happens will be followed. These studies are important because they will show how proteins avoid misfolding, which could compromise their biological function.

This project will promote participation of underrepresented graduate and undergraduate students and will be a benchmark to learn advanced biological techniques and mechanisms. Classroom demonstrations involving colorful renderings of protein folding/unfolding will be developed, providing unprecedented opportunities for active learning.

This project focuses on the earliest stages of protein folding as nascent chains emerge out of the ribosome in the absence and presence of molecular chaperones. Very little is still known about how proteins fold in the cellular environment. Importantly, translation through the ribosome is often required to generate fully folded and bioactive proteins within the in vivo milieu.

Yet, the role of the ribosome in the protein-making process is still extremely poorly understood. In this research, single-domain proteins of variable size originating from codon-optimized genes in E. coli will be studied. The project involves the determination of 3D structure of ribosome-bound nascent proteins by single-particle cryo-electron microscopy, as well as the elucidation of nascent-protein dynamics by time-resolved fluorescence anisotropy.

In addition, nascent-protein ribosome interactions will be studied by chemical crosslinking. Through the proposed studies, insights into how interactions with the ribosome and molecular chaperones ensure that nascent proteins attain their native state devoid of competing aggregation will be gained. The principal investigator and her research group has determined that the last stages of translation and the events accompanying full-length ribosome-bound nascent-chain (RNC) release from the ribosome are crucial to kinetically channel proteins to their native state, away from aggregation.

Next, these stages at much higher resolution will be characterized to fully understand the function of ribosome and molecular chaperones in the cellular context. These investigations are significant for a better understanding of biological phenomena as well as for the future custom-design of proteins able to more robustly and efficiently withstand severe environmental perturbations.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.