Successful protein folding involves the conversion of a linear polypeptide into a stable and biologically active 3D structure. This process has been studied for decades by denaturing purified, full-length polypeptides in vitro, diluting away the denaturant, and observing the refolding process. What is still lacking, however, is an understanding of how these in vitro results relate to protein folding in vivo. The initiation of protein folding in vivo is fundamentally different: the starting ensemble for folding in vivo is a growing nascent polypeptide chain, rather than a full-length chain. We have very little information about how growth of the nascent chain affects the energy landscape for folding. Yet this difference may help explain why some native state topologies are well represented in vivo, but challenging to refold in vitro as full-length polypeptide chains. In this proposal, the influence of translation on protein folding will be investigated from three unique perspectives: (1) Early events: nascent chain diffusion and collapse. Our collaborator Lisa Lapidus has recently shown that intramolecular diffusion in vitro is extremely slow for an unfolded, collapsed protein in buffer, relative to high concentrations of denaturant. We have adapted Lapidus's methods to determine the extent to which a ribosome-bound nascent chain can collapse, and the intramolecular diffusion rate for that collapsed state, as these parameters will affect the size and shape of the energy landscape accessible to that nascent chain after its release from the ribosome. (2) Synonymous rare codons reduce local translation rate, which can alter co-translational folding mechanisms, presumably by altering the accessible energy landscape for folding. Remarkably, recent results emerging from our lab and others now indicate that, for some proteins, local translation rate can also alter the native structure, enabling access to an energy minimum not accessible under other translation rate patterns. We will measure the capacity of synonymous codon selection (mRNA sequence) to modify protein structure, thereby characterizing its modifications to the energy landscape for folding in vivo. (3) In the first project period, we showed that synonymous rare codons are not randomly distributed along gene sequences, but tend to cluster together, despite established negative effects of such clustering (including reduced translation rate). What positive cellular effects might overcome these known negatives? Certainly modified co-translational folding (to increase the yield of correctly folded protein) is one effect, but broader effects, including impact on other cell functions, must also be considered. We have developed a tunable in vivo system with which to answer previously unanswerable questions regarding the effects of co-translational folding on cell physiology, including cell fitness. Taken together, results from this proposal will reveal how translation modifies early folding events, native protein structures, and cell physiology. Several decades of in vitro refolding studies have revealed general principles for protein refolding in the test tube;results from this proposal will be used to develop principles for protein folding in vivo.

Public Health Relevance

Proteins are synthesized as linear strings of amino acids, but typically fold up into a three-dimensional shape in order to function. Decades of research have illuminated how full-length proteins fold in test tubes, but we still know virtually nothing about how proteins fold in the cellular environment. The proposed research will determine how the earliest cellular folding events, which occur while the protein chains are still undergoing synthesis by the ribosome, can affect the protein folding mechanism, the native protein structure and the overall fitness of the cell.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074807-09
Application #
8640188
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
2005-09-01
Project End
2015-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
9
Fiscal Year
2014
Total Cost
$290,183
Indirect Cost
$82,062
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Sander, Ian M; Chaney, Julie L; Clark, Patricia L (2014) Expanding Anfinsen's principle: contributions of synonymous codon selection to rational protein design. J Am Chem Soc 136:858-61
Braselmann, Esther; Chaney, Julie L; Clark, Patricia L (2013) Folding the proteome. Trends Biochem Sci 38:337-44
Ugrinov, Krastyu G; Clark, Patricia L (2010) Cotranslational folding increases GFP folding yield. Biophys J 98:1312-20
Clarke 4th, Thomas F; Clark, Patricia L (2010) Increased incidence of rare codon clusters at 5' and 3' gene termini: implications for function. BMC Genomics 11:118
Evans, Michael S; Sander, Ian M; Clark, Patricia L (2008) Cotranslational folding promotes beta-helix formation and avoids aggregation in vivo. J Mol Biol 383:683-92
Clarke 4th, Thomas F; Clark, Patricia L (2008) Rare codons cluster. PLoS One 3:e3412
Kutrowska, Beata Wielgus; Narczyk, Marta; Buszko, Anna et al. (2007) Folding and unfolding of a non-fluorescent mutant of green fluorescent protein. J Phys Condens Matter :285223