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A comparison of the folding characteristics of free and ribosome‐tethered polypeptide chains using limited proteolysis and mass spectrometry
Author(s) -
Rajabi Khadijeh,
Reuther Julia,
Deuerling Elke,
Radford Sheena E.,
Ashcroft Alison E.
Publication year - 2015
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2702
Subject(s) - proteolysis , chemistry , ribosome , protein folding , mass spectrometry , folding (dsp implementation) , biochemistry , biophysics , rna , enzyme , biology , chromatography , electrical engineering , gene , engineering
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.