The mechanism of protein folding has been the subject of intense research over the past several decades. Most studies are carried out on full-length proteins in solution. Although these studies are informative, they do not give a complete picture of how proteins fold in cells as they are synthesized by the ribosome. Such cotranslational folding is poorly understood, as is the potential role of the ribosome in directing proper folding of proteins.

“It has been long suggested that cotranslational folding differs from the spontaneous folding-unfolding of proteins in solution, because the narrow polypeptide exit tunnel of the ribosome restricts the choice of potential conformations for folding,” notes Marina Rodnina of the Max Planck Institute for Biophysical Chemistry. To better understand these differences, she and lab members Wolf Holtkamp, a postdoctoral fellow, and Goran Kokic, an undergraduate student, developed methods to monitor a protein's structure as it is synthesized by the ribosome.

For their experiments, the team used a purified Escherichia coli in vitro translation system to translate a 112–amino acid protein consisting of an α-helical domain and linker. They then studied the folding of the emerging, or nascent, protein using fluorescence resonance energy transfer (FRET), which can measure the distance between two fluorophores. They generated synchronized initiated translating complexes in which the first amino acid was labeled with a fluorophore. They then allowed translation to proceed by adding in the necessary factors. For site-specific labeling of an internal position in the nascent protein, they included a lysine tRNA charged with a fluorescently labeled lysine derivative.

The team then obtained FRET measurements to examine the effects of the extent of translation on the structure of the protein. The team expected that folding would be hindered in short versions of the protein because of confinement by the polypeptide exit channel, and as expected, when only 41 amino acids of the protein were translated, there was low FRET signal, demonstrating that the protein was held in an extended form by the exit channel. Conversely, when the full-length protein was synthesized such that the entire α-helical domain was free from the exit channel, a FRET signal consistent with the folded protein was observed. Interestingly, for intermediate lengths of the protein, a high FRET signal was measured that was distinct from the signals of both the folded protein and the extended form, indicating that the nascent peptide formed a unique compact structure.

These results were corroborated by further experiments using mutants of the protein with disrupted folding and by studies using photoinduced electron transfer between the first labeled amino acid and engineered tryptophan residues, a technique that is very useful for probing structures at short length scales. These experiments provided evidence complementary to that from the team's FRET studies and confirmed that the protein folded into a compact structure distinct from the final folded structure after leaving the exit channel, a result that Rodnina recalls was “most surprising.”

These experiments provide important evidence that the ribosome can affect the structure of translated proteins prior to final folding. The ribosome could have the potential to prevent misfolding or even direct proper folding of proteins in the complex cellular milieu by inducing or stabilizing folding intermediates, although future work will be necessary to further elucidate its role. Overall, the future is bright for these complementary approaches, and Rodnina hopes to use them to study how other protein domains fold, as well as to examine how other factors, such as chaperones, affect protein folding on the ribosome.