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. Careful measurements of refolding kinetics and populated partially folded conformations have revealed much about the refolding process. What is still lacking, however, is an understanding of how these in vitro results relate to protein folding in vivo. Protein folding in vivo is a fundamentally different process: In particular, 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 this fundamentally different landscape for the growing chain relates to refolding mechanisms observed in vitro. Yet this difference may help explain why there are some native state topologies that are well represented in vivo, but difficult to refold in vitro. In this proposal, the influence of translation on protein folding in vivo will be studied from two perspectives: (1) The effects of translation pausing on folding yield. Rare codons can cause the ribosome to pause during translation, and literature reports have connected pauses with folding yield. For this proposal, novel methods have been developed to identify and modulate rare codon-derived translation pauses, and the effects on downstream folding. (2) The relationships between co-translational nascent chain conformations, in vitro refolding conformations, and the conformations of free, truncated polypeptides. Does protein refolding in vitro faithfully reproduce the conformations of nascent chains? For native structures, with contacts between distant portions of the polypeptide chain, this is unlikely. What alternative conformations are formed by these nascent chains? Are they also formed by free peptides? Results from in vitro refolding studies have revealed general principles for protein refolding; results from this proposal will be used to develop principles for protein folding in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074807-04
Application #
7492938
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
2005-09-01
Project End
2010-08-31
Budget Start
2008-09-01
Budget End
2009-08-31
Support Year
4
Fiscal Year
2008
Total Cost
$270,232
Indirect Cost
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
Chaney, Julie L; Steele, Aaron; Carmichael, Rory et al. (2017) Widespread position-specific conservation of synonymous rare codons within coding sequences. PLoS Comput Biol 13:e1005531
Faisal, Fazle E; Newaz, Khalique; Chaney, Julie L et al. (2017) GRAFENE: Graphlet-based alignment-free network approach integrates 3D structural and sequence (residue order) data to improve protein structural comparison. Sci Rep 7:14890
Jacobson, Giselle N; Clark, Patricia L (2016) Quality over quantity: optimizing co-translational protein folding with non-'optimal' synonymous codons. Curr Opin Struct Biol 38:102-10
Clark, Patricia L (2016) How to Build a Complex, Functional Propeller Protein, From Parts. Trends Biochem Sci 41:290-292
Brodsky, Jeffrey L; Clark, Patricia L (2014) Protein folding in the cell, from atom to organism. FASEB J 28:5034-8
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
Clark, Patricia L; Ugrinov, Krastyu G (2009) Measuring cotranslational folding of nascent polypeptide chains on ribosomes. Methods Enzymol 466:567-90

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