Chain dynamics of nascent polypeptides emerging from the ribosome.

Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.

Experimental and computational analysis of translation products in apomyoglobin expression.

This work focuses on the experimental analysis of the time-course of protein expression in a cell-free system, in conjunction with the development of a computational model, denoted as progressive chain buildup (PCB), able to simulate translation kinetics and product formation as a function of starting reactant concentrations. Translation of the gene encoding the apomyoglobin (apoMb) model protein was monitored in an Escherichia coli cell-free system under different experimental conditions. Experimentally observed protein expression yields, product accumulation time-course and expression completion times match with the predictions by the PCB model. This algorithm regards elementary single-residue elongations as apparent second-order events and it accounts for aminoacyl-tRNA regeneration during translation. We have used this computational approach to model full-length protein expression and to explore the kinetic behavior of incomplete chains generated during protein biosynthesis. Most of the observed incomplete chains are non-obligatory dead-end species, in that their formation is not mandatory for full-length protein expression, and that they are unable to convert to the expected final translation product. These truncated polypeptides do not arise from post-translational degradation of full-length protein, but from a distinct subpopulation of chains which expresses intrinsically more slowly than the population leading to full-length product. The PCB model is a valuable tool to predict full-length and incomplete chain populations and formulate experimentally testable hypotheses on their origin. PCB simulations are applicable to E.coli cell-free expression systems (both in batch and dialysis mode) under the control of T7 RNA polymerase and to other environments where transcription and translation can be regarded as kinetically decoupled.

In vitro expression and characterization of native apomyoglobin under low molecular crowding conditions.

The labile nature of membranes and organelles poses serious challenges to in situ biomolecule characterization in intact cells. Cell-free in vitro systems provide an alternative promising medium for the expression and characterization of protein conformation and function in a biochemical context that bears several similarities to the cellular environment. In addition, cell-free transcription-translation has recently emerged as a convenient method for protein selective isotope labeling, providing significant advantages for detailed NMR analysis. We report the cell-free expression of the model protein apomyoglobin (apoMb) in an Escherichia coli cell-free system and the effect of polyethylene glycol (PEG) on the expression yields. In contrast with in vivo protein production under control of the strong T7 promoter, apoMb is expressed in vitro in 100% soluble form. In-gel tryptic digestion followed by mass spectrometry were performed to confirm the protein identity. In order to probe the conformation of the newly expressed protein and investigate the feasibility of in situ structural analysis, high resolution protein characterization was carried out by 2D NMR spectroscopy. In vitro apoMb expression in a PEG-free environment is a convenient method for the production of soluble native-like protein under conditions amenable to selective isotopic labeling. Yields can be easily scaled-up by dialysis-assisted cell-free expression.