Tajima, T; (2015) Folding during ribosomal biosynthesis of yellow fluorescent protein: Investigations by fluorescence spectroscopy and NMR spectroscopy. Doctoral thesis , UCL (University College London).

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Abstract

During biosynthesis on the ribosome, the nascent polypeptide chain emerges in a vectorial fashion, one amino acid at a time and it has its first opportunity to sample conformational space. Study of structural and molecular biology has shown that nascent chains (NC) can co-translationally acquire its native structure, however how an emerging NC forms the native conformation is a fundamental question in modern biology. Yellow fluorescent protein (YFP), a red shifted yellow emission variant of green fluorescent protein (GFP) that is consisted of 238 amino acid residues is highly distinct because of its fast maturation of fluorophore and its rapid folding kinetics. In addition, YFP has been extensively studied using X-ray crystallography, nuclear magnetic resonance (NMR), and biophysics, which make it an ideal system for examining protein folding on the ribosome. In this study, therefore, we created a series of ribosome-nascent chain complex (RNC) of YFP to simulate the progressive emergence of NC. We also developed an in vitro transcription translation system to generate isotopically-labelled RNCs for both biophysics and NMR spectroscopy. To complement the RNC study, we also created a co-translational folding mimetic using C-terminal truncations of isolated YFP to evaluate folding in vitro. Using these approaches, the co-translational folding of YFP was observed using both spectroscopies of fluorescence and NMR at a residue specific level. Here we demonstrate that native folding can take place very close to the ribosome in which YFP is separated by 19 amino acids from the ribosome’s peptidyl transferase centre (PTC), and that YFP can tolerate the absence of up to 14 amino acids from its C-terminus and still acquire a native fold.

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