Many large, multi-domain proteins are inherently prone to misfolding and aggregation. As soon as they emerge from the ribosome during translation, they are met by molecular chaperones that help them fold into their native structures. While it is clear that molecular chaperones are essential for correct folding in vivo, we know very little about the underlying mechanisms. The long-term goal of our studies is to define the folding mechanisms of complex, multi-domain proteins in the cell.
The aim of this proposal is to determine the function of two nascent chain-binding chaperones, Trigger factor and the DnaK system, in the folding of a model multi-domain protein, EF-G. We will study how these chaperones guide the folding of nascent multi- domain proteins using single-molecule force spectroscopy with optical tweezers. This approach is uniquely suited to manipulate and observe the folding of individual nascent polypeptides in the complex environment of the ribosome and molecular chaperones. We will first measure folding transitions of nascent proteins on the ribosome to define their folding energy landscapes. Then, we will determine how Trigger factor and DnaK change these energy landscapes. Our in vitro experiments will reveal in mechanistic detail and with single- molecule resolution how these chaperones contribute to efficient folding. In the cell, the nascent polypeptide interacts with a network of molecular chaperones and other factors that influence its folding and processing. To complement our single-molecule experiments, we will carry out experiments in live cells to determine folding waypoints of multi-domain proteins in vivo. In addition, we will define how chaperones engage their substrates in living cells. Together, these studies will establish a framework for mechanistically understanding protein folding in vivo. Protein folding is of key importance for cellular protein homeostasis. Protein misfolding and aggregation are a hallmark of many diseases, including neurodegenerative diseases and cancer. The research proposed here may ultimately lead to a better understanding and possible treatments for these diseases.

Public Health Relevance

In all organisms, proteins must ?fold? into well-defined three-dimensional structures, often with the help of molecules called molecular chaperones, in order to function properly. Defects in folding have severe consequences and can lead to neurodegenerative disorders and cancer, but how this misfolding is avoided in healthy cells is poorly understood. The proposed research will investigate mechanisms of protein folding and molecular chaperone function. Successful completion of this proposal promises to provide insights into novel therapeutic strategies for human diseases that involve misfolded proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Phillips, Andre W
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Johns Hopkins University
Schools of Arts and Sciences
United States
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Kaiser, Christian M; Liu, Kaixian (2018) Folding up and Moving on-Nascent Protein Folding on the Ribosome. J Mol Biol 430:4580-4591
Liu, Kaixian; Rehfus, Joseph E; Mattson, Elliot et al. (2017) The ribosome destabilizes native and non-native structures in a nascent multidomain protein. Protein Sci 26:1439-1451