Translation and folding of single proteins in real time

Abstract

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

Synthesis runs counter to directional folding of a nascent protein domain

“…Our studies provide an example of folding occurring in the direction opposite to that of synthesis and thus contrasts with previous findings of gradual compaction and folding concomitant with protein elongation 48 . Once the full G-domain has emerged from the ribosome, folding occurs in several steps that appear similar on the ribosome and in isolation ( Figure 6A).…”

Sequence and structural patterns detected in entangled proteins reveal the importance of co-translational folding

“…In the context of knotted proteins, single molecule force spectroscopy techniques were shown to be particularly useful in controlling the topology of the unfolded state [46]. Similarly, both "in vivo" folding experiments [47] and appropriate simulation protocols [48][49][50] could be employed to test the possible role of cotranslational folding in determining the patterns detected for entangled motifs: double cysteine mutants would then be predicted to be more deleterious for the folding of C-terminal threads with respect to N-terminal threads. In all cases, it is essential to gather statistics over several different proteins before validating or rejecting our hypothesis; the signals that we reveal in this contribution are statistical in nature; therefore we do not expect all entangled loops to form late in the folding process nor all C-terminal threads to be cotranslationally disfavored.…”

Co-translation drives the assembly of mammalian nuclear multisubunit complexes

“…These mechanisms could play an important role in maintaining cellular health as excess orphan protein subunits can overburden protein folding and quality control machineries 35 . There is a strong correlation between the amino acid sequence of a protein, its translation rate and co-translational folding 36 . Rare codons in the mRNAs decrease the rate of translation, thereby allowing the protein to fold co-translationally 32 .…”