Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Probing Protein Folding on the Ribosome by Solution State NMR Spectroscopy
The means by which a polypeptide chain acquires its unique three dimensional structure is a fundamental question in biology. During its synthesis on the ribosome, a nascent chain emerges in a vectorial manner and will begin to fold in a co-translational fashion (1, 2). Our current knowledge of protein folding at the level of individual residues has come overwhelmingly from a combination of computer simulations and experimental studies of protein denaturation and renaturation in vitro, using biochemical and biophysical methods.
To account for the contribution of the protein translation machinery, namely the ribosome, to the de novo folding of a nascent polypeptide chain, we have recently developed a protocol combining cell-free synthesis, selectively isotope labeling and rapid multidimensional heteronuclear NMR spectroscopy to identify the presence of a well-folded protein domain structure in part of the nascent chain as a ternary peptido-tRNA, ribosome complex (3). This has demonstrated the feasibility of NMR studies on supra-biomolecular complexes such as the ribosome-nascent chain complex at its functional states. Residue-specific analysis shows that the dynamics of a co-localized region in the folded domain of the nascent chain is strongly affected in the ternary complex, suggesting transient interactions between the ribosome and this part of the folded domain.
Intriguingly, our recent data suggest that the ribosome attachment has different effects on the internal dynamics of the folded domain at the backbone and side-chain levels. These findings represent a first step towards a description in atomic detail of the process of protein folding coupled to translation of the genetic code. Recent developments in further systematic characterization of the chain length-dependent nascent chain folding will also be discussed, including some evidence of the existence of a distinct folding intermediate of a ribosome bound nascent chain in contrast to the highly cooperative urea-induced unfolding process of the same construct in isolation.
References and Footnotes
Shang-Te Danny Hsu1,*
1Department of Chemistry