Structural and Mechanstic Aspects of Cotranslational Protein Folding

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

TITLE: Structural and Mechanistic Aspects of Cotranslational Protein Folding This project will lead to a better understanding of how proteins attain their 3-dimensional shape in the cell, providing a gateway to a deeper understanding and control of crucial life-related processes. The shape of proteins is extraordinarily important because it determines biological activity. However, little is known about how proteins achieve their bioactive three-dimensional shape inside the living cell. This research will elucidate how proteins gain their precise fold from the earliest stages of their life, from the time they are made a by a complex machine known as the ribosome. The role of the ribosome and molecular helpers known as chaperones will be explored. Molecular movies, showing folding as a function of time, will be generated to watch the microscopic details of the folding process in real time. This research will foster the participation of several underrepresented graduate students, and will be a benchmark to learn advanced biophysical techniques as well as the mechanisms of protein folding in the cell. In addition, classroom demonstrations showing illustrative renderings of the protein folding and unfolding processes will be developed, providing unprecedented opportunities for active learning and for undergraduate student participation in the classroom. As part of the broader impacts of this project, campus pre-visits for minority and disadvantaged undergraduate students will also be organized, to encourage them to pursue graduate studies. Despite the extensive literature available on the folding of purified proteins upon dilution from denaturant and upon temperature-jump, there is very little information on how proteins fold in the cellular environment, where chaperones, molecular crowding and the ribosome can profoundly affect the sequence and nature of the events leading to bioactive proteins. This project addresses the earliest stages of folding in the cellular context, as proteins emerge from the ribosome. The role of nascent chain compaction and thermodynamic stability, together with the effect of the ribosomal surface and the rate of full-length protein release from the ribosome, will be addressed at the molecular level. Experiments will be carried out by a combination of molecular-biology and biophysical techniques, including fluorescence depolarization and pulsed proteolysis on wild-type and rationally designed variants of the E. coli globin apoHmpH and on other control nascent proteins. Data will be collected in the absence and presence of the DnaK/DnaJ/GrpE chaperones, to assess the role played by each of these folding facilitators. The specific interaction between the ribosomal surface and nascent proteins will be assessed as function of ionic strength. The rate of release of ribosome-bound nascent proteins will be followed in real time to gain high-resolution insights into the events accompanying protein birth. Overall, this project will lead to an improved understanding of intracellular protein folding, one of the most fascinating aspects of gene expression.