Biophysical Aspects of Co- and Immediately Post-Translational Protein Folding

Proteins are important components of living organisms. They perform crucial biological functions. They enable, support and protect key aspects of our daily life, including seeing objects, hearing sounds, talking, thinking, and walking. The three-dimensional shape of proteins is extraordinarily important because it determines biological activity. The goal of this research is to reveal how proteins achieve their complex and highly organized three-dimensional shape within living cells. This process, called protein folding, resembles folding laundry. Yet, it is much more complex than that, and it occurs at the level of individual protein molecules in the complex environment of live cells. To date, we know very little about how proteins fold in biological media. This is unfortunate because improperly folded proteins cannot perform their biological function. Therefore, protein misfolding leads to important challenges in biotechnology and pharmaceutical sciences. In addition, incorrectly folded proteins are related to deadly human diseases like ALS and Huntington’s disease. To understand the mechanisms of protein folding, this project will elucidate the high-resolution structure and dynamic motions of the ribosome, the protein-making machinery, in the presence of nascent proteins, as they fold. With the help of sophisticated lasers, magnets and electron beams, we will reveal how nascent proteins interact with the ribosome, and how the ribosome, and other interacting partners, help proteins fold. We will also follow protein making in real time and watch how the protein obtains its shape as it happens, akin to taking a three-dimensional movie. These studies are important because they will provide key training for graduate and undergraduate students interested in joining the biotechnology workforce. In addition, this research will reveal how proteins can be induced to always reach their correct fold. This will enable improving the yields and efficiency of protein production, providing a gateway to generate cheaper custom-shaped biomaterials and pharmaceuticals that are crucial for our national manufacturing goals. This project involves the determination of 3D structure and dynamics of ribosome-bound nascent proteins via a combination of single-particle cryo-electron microscopy and time-resolved fluorescence anisotropy. In addition, noncovalent contacts involving nascent proteins will be explored by chemical crosslinking. Through the proposed studies, we will gain insights into how interactions with the ribosome, molecular chaperones and cofactors ensure that nascent proteins attain their native state devoid of competing aggregation. Importantly, the last stages of translation are crucial to kinetically channel proteins to their native state, away from aggregation. These stages will also be characterized in real time. The above investigations are significant for student training, for a better understanding of biological phenomena, and for the custom-design of proteins able to respond to custom stimuli and robustly withstand 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. NSF Award ID: 2547879 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Silvia Cavagnero | Institution: University of Wisconsin-Madison, MADISON, WI | Award Amount: $914,922 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2547879 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2547879.html