De novo protein folding is the fundamental process by which a polypeptide chain acquires its native conformation for the first time. All proteins are synthesised by the ribosome and can begin to fold co-translationally as the nascent chain emerges vectorially from the ribosome exit tunnel, exploring an evolving energy landscape shaped by translation kinetics, the ribosome and molecular chaperones. Understanding how a protein reaches its native state therefore requires resolving folding both structurally and kinetically, in real time, as it happens on the ribosome.
This project will establish single-molecule Förster resonance energy transfer (smFRET) to monitor co-translational folding in real time, bridging structural detail with kinetic resolution. The project will establish an in vitro translation system that enables ribosome immobilisation, site-specific labelling of the ribosomal subunits and co-translational incorporation of photostable fluorophores into the nascent chain through genetic code expansion. Extensive FRET coverage across multiple labelling positions will enable ab initio structure determination of the nascent chain conformations, while ribosome ratcheting will report on codon-level translation kinetics.
Using a specialised, single-photon counting confocal microscope capable of multiplexed detection, fluorescence correlation spectroscopy and fluorescence lifetime imaging, conformational exchange will be captured across the nanosecond-to-second regime. FRET-derived distance distributions will then be integrated with molecular dynamics simulations to reconstruct an atomistic movie of nascent-chain folding in real time. The student will gain interdisciplinary training in single-molecule fluorescence, in vitro translation, protein biochemistry and structural biology, and will work alongside computational colleagues to integrate the experimental data with molecular dynamics simulations.