title: A comparison of the folding characteristics of free and ribosome-tethered polypeptide chains using limited proteolysis and mass spectrometry.
abstract: The kinetics and thermodynamics of protein folding are commonly studied in vitro by denaturing/renaturing intact protein sequences. How these folding mechanisms relate to de novo folding that occurs as the nascent polypeptide emerges from the ribosome is much less well understood. Here, we have employed limited proteolysis followed by mass spectrometry analyses to compare directly free and ribosome-tethered polypeptide chains of the Src-homology 3 (SH3) domain and its unfolded variant, SH3-m10. The disordered variant was found to undergo faster proteolysis than SH3. Furthermore, the trypsin cleavage patterns observed show minor, but significant, differences for the free and ribosome-bound nascent chains, with significantly fewer tryptic peptides detected in the presence of ribosome. The results highlight the utility of limited proteolysis coupled with mass spectrometry for the structural analysis of these complex systems, and pave the way for detailed future analyses by combining this technique with chemical labeling methods (for example, hydrogen-deuterium exchange, photochemical oxidation) to analyze protein folding in real time, including in the presence of additional ribosome-associated factors.
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Khadijeh
authors.initials: K
authors.lastname: Rajabi
authors.affiliation: Department of Biology, University of Konstanz, 78457, Konstanz, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Reuther
authors.affiliation: Department of Biology, University of Konstanz, 78457, Konstanz, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Deuerling
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D058767:Protein Unfolding; D059748:Proteolysis; D012270:Ribosomes / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D018909:src Homology Domains
chemical_list: D010455:Peptides
keywords: Src-homology 3; circular dichroism spectroscopy; limited proteolysis; nanoelectrospray ionisation-mass spectrometry; ribosome-nascent chain complexes; trypsin digest