Karyadi, ME;
(2013)
Characterisation of a model of a co-translational folding energy landscape.
Doctoral thesis , UCL (University College London).
Abstract
Co-translational protein folding is the de novo folding which occurs vectorially during polypeptide synthesis while the C-terminus is still tethered to the ribosome. In this work, we have created a model of co-translational protein folding in vitro by preparing and characterising a series of nine progressive truncations from the C-terminus of the immunoglobulin domain ddFln5, to mimic the progressive emergence of the nascent chain (NC) synthesis as it occurs on the ribosome. Using Nuclear Magnetic Resonance (NMR) spectroscopy, in combination with other biophysical methods we have mapped, at multiple temperatures, the emergence of native-like folded structure from the denatured state through a series of three intermediate and increasingly native-like states. The backbone chemical shifts of all states have been assigned using standard triple-resonance methods. The backbone dynamics of the intermediate states have also been investigated using 15N relaxation measurements revealing overall native-like dynamics. Furthermore, as a complement to the study of the isolated protein we have developed an in vivo methodology to express and purify ddFln5 on the ribosome stalled via a secretion monitor (SecM) motif to obtain ribosome nascent chain complexes (RNCs) for studies using NMR.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Characterisation of a model of a co-translational folding energy landscape |
| Language: | English |
| Additional information: | Permission for digitisation not received. |
| UCL classification: | UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1396246 |
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