Co-translational mechanisms of protein folding studied on the single molecule level
Biochemistry
Final Report Abstract
Co-translational protein folding is coupled to protein synthesis and is mediated by spatial constraints and interactions within as well as outside the ribosomal tunnel. However, it is still not yet entirely understood how synthesis (i.e. polypeptide chain elongation) and folding cooperate to accomplish a fully functional protein. Our previous work provided us the required technical knowledge and experience to bring together force- and fluorescence-based single molecule techniques in order to achieve a deeper understanding of this interconnection. First, we conducted a study to understand in more depth the co-translational folding of a small protein domain (ADR1a) inside the ribosomal tunnel combining smFRET and optical tweezers. Here electrostatic interactions between the protein and ribosome seem to contribute to the observed folding acceleration and stabilization of ADR1a. Next, we tried to apply this approach to the study of a larger protein, namely the two-domain protein phosphoglycerate kinase from yeast (yPGK). To this end, folding transitions of the entire protein were first characterized with smFRET and properties of the transitions with regard to the underlying domain topology were analysed. The approach to make use of cell free synthesized dual dye-labelled polypeptide chains in order to perform smFRET studies for characterizing nascent yPGK chains of different length, corresponding to different stages of synthesis and folding, could not be realised yet. However, an additional approach employing cryo-EM with single particle analysis of ribosome nascent chain complexes (RNCs) showed first promising results. Finally, the attempt to monitor in real time the synthesis and folding of yPGK under force (same approach that was carried out with ADR1a) has been started, but could not be completed successfully due to problems with the cell free synthesized yPGK chains.
Publications
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The ribosome modulates folding inside the ribosomal exit tunnel. Communications Biology, 4(1).
Wruck, Florian; Tian, Pengfei; Kudva, Renuka; Best, Robert B.; von Heijne, Gunnar; Tans, Sander J. & Katranidis, Alexandros
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Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution. Biomolecules, 12(3), 468.
Yukhnovets, Olessya; Höfig, Henning; Bustorff, Nuno; Katranidis, Alexandros & Fitter, Jörg
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“Snapshots of co-translational multi-domain protein folding”, 66th Annual Meeting of the Biophysical-Society 2022, Biophys. J. 121 (3), 284A-284A
N. Bustorff & A. Katranidis, J. Fitter
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Features of Protein Unfolding Transitions and Their Relation to Domain Topology Probed by Single-Molecule FRET. Biomolecules, 13(9), 1280.
Bustorff, Nuno & Fitter, Jörg
