Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy

Abstract

The means by which a polypeptide chain acquires its unique 3-D structure is a fundamental question in biology. During its synthesis on the ribosome, a nascent chain (NC) emerges vectorially and will begin to fold in a cotranslational fashion. The complex environment of the cell, coupled with the gradual emergence of the ribosome-tethered NC during its synthesis, imposes conformational restraints on its folding landscape that differ from those placed on an isolated protein when stimulated to fold following denaturation in solution. To begin to examine cotranslational folding as it would occur within a cell, we produce highly selective, isotopically labeled NCs bound to isotopically silent ribosomes in vivo. We then apply NMR spectroscopy to study, at a residue specific level, the conformation of NCs consisting of different fractional lengths of the polypeptide chain corresponding to a given protein. This combined approach provides a powerful means of generating a series of snapshots of the folding of the NC as it emerges from the ribosome. Application of this strategy to the NMR analysis of the progressive synthesis of an Ig-like domain reveals the existence of a partially folded ribosome-bound species that is likely to represent an intermediate species populated during the cotranslational folding process.

Fig. 1.

The 70S ribosome and RNC constructs. Left: schematic diagram of the 70S ribosome with the 30S and 50S subunits highlighted in red and blue, respectively. The PTC is shown occupied by tRNA (purple). Mapped onto the ribosome is the NC construct as produced via the pLDC vectors. From the C terminus at the PTC is the 17-residue SecM motif, (yellow), followed by the selected NC (blue). The NC is shown fused to a hexa-His tag; Center: the general features of the pLDC-17 vector are shown (above) in the N to C direction. Schematics of the two RNCs described in this study are shown below: ddFLN_646–839-RNC is Dom5 plus a 90 residue sequence derived from Dom6 fused to SecM; ddFLN_646–750-RNC is Dom5 alone fused to SecM; Right: representation of the two domain structure (from 1QFH.pdb) of Dom 5 and Dom 6 (ddFLN_646–857). The ddFLN_646–839-RNC vector comprises an NC construct designed such that the final strand in Dom6 (G-strand, shaded gray) is absent.

Fig. 2.

Generation of RNCs in vivo using E. coli expression. After transformation of the NC-containing pLDC-17 vector into E. coli, cell growth to high cell densities in MDG medium was allowed for up to 20 h, 37 °C, 280 rpm. Cells were then harvested and resuspended in M9 expression medium in the absence of a nitrogen source. The nitrogen source was depleted before the addition of ¹⁵N ammonium chloride and expression induced at 25 °C. Cells were harvested 45 min after induction.

Fig. 3.

Purification scheme for RNCs. RNCs were purified using a two-step purification process. After expression, the E. coli cells were lysed using a French press, the lysate applied to a Co²⁺ affinity column, and the eluate subjected to a 10–35% (vol/vol) sucrose gradient. After fractionation the RNCs were analyzed on SDS/PAGE (silver stain shown) and probed with an anti-His antibody to confirm the presence of the NC (shown by the arrow).

Fig. 4.

Comparison of ¹⁵N-¹H correlation NMR spectra of ddFLN_646–839-RNC and ddFLN_646–750-RNC with that of isolated Dom5. A selection of cross-peaks has been highlighted (red circles) in each of the three ¹⁵N-¹H correlation spectra for ready comparison. Cross-peaks in the spectrum of ddFLN_646–839-RNC show similarities to cross-peaks present in spectra of the isolated Dom5 protein under native conditions (0 M urea), indicating that, on the ribosome, the in vivo generated NC is able to acquire native-like structure. ddFLN_646–750-RNC_, by contrast, shows a predominantly unfolded spectrum, as compared to isolated Dom5 in denaturing conditions (8 M urea) although there are nine well-resolved cross-peaks corresponding to the signals of several residues present in the isolated Dom5 spectrum under native conditions (0 M urea); the latter residues are indicative of the presence of native-like structure. The spectrum of ddFLN_646–750-RNC therefore is neither that of a fully folded or of a fully unfolded protein.

Fig. 5.

Comparison of ¹³C-¹H NMR spectra of ddFLN_646–839-RNC and ddFLN_646–750-RNC with that of isolated Dom5 (ddFLN_646–839). The ¹H-¹³C methyl region spectra of the two RNCs, ddFLN_646–839 (Middle) and ddFLN_646–750 (Bottom), are compared to that (Top) of native ddFLN_646–839 (0 M urea). Highlighted in each spectrum is a selection of well resolved cross-peaks whose chemical shifts coincide with native-like structure (within 0.05 ppm in the ¹H dimension in isolated Dom5). The long RNC (ddFLN_646–839-RNC) shows the equivalent cross-peak dispersion as seen for Dom5, while the short RNC, ddFLN_646–750-RNC, shows several dispersed cross-peaks (corresponding to V664, V717, L733, and V729 in the fully folded domain and clearly separated from random coil chemical shift values), coinciding with native-like structure but the absence of the most high field shifted methyl groups (those between −0.1 and −0.5 ppm in the ¹H dimension) indicates the presence of a partially folded structure.

Fig. 6.

The structure of Dom5 with residues labeled that appear to have crosspeaks showing native-like structure in ddFLN_646–750. Mapped on to structural models (from 1QFH.pdb) of Dom5 are residues with cross-peaks in the ¹⁵N-¹H spectra (blue spheres) and cross-peaks in the ¹³C-¹H (red spheres) spectra of Dom5 that are within 0.1 and 0.05 ppm (¹H dimension) respectively of cross-peaks in both (A) ddFLN_646–839-RNCand (B) ddFLN_646–750-RNC. Shown in purple spheres are cross-peaks that are visible in both the ¹⁵N-¹H and ¹³C-¹H spectra. In the case of ddFLN_646–750-RNC, the residues of the final G-strand of Dom5 remains within the exit tunnel although cross-peaks are, however, observed in the NMR spectrum of ddFLN_646–750-RNC (lower left spectrum in Fig. 4 and lower spectrum in Fig. 5) that could correspond to native-like structure in other regions including strand B (the peak attributed to I674 is visible in ¹⁵N spectra and that attributed to A670 in ¹³C spectra, respectively), strand E (V717 visible in ¹³C spectrum), and strand F (peaks for N728 and N730 are visible in ¹⁵N spectra, and those for V729 and L733 can be seen in¹³C spectra) and also several connecting loop regions.