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. 2009 Oct;18(10):2003-15.
doi: 10.1002/pro.196.

Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

Jamie P Ellis  1 , Peter H Culviner, Silvia Cavagnero
Affiliations

Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

Jamie P Ellis et al. Protein Sci. 2009 Oct.

Erratum in

  • Protein Sci. 2010 Aug;19(8):1600

Abstract

We still know very little about how proteins achieve their native three-dimensional structure in vitro and in the cell. Folding studies as proteins emerge from the mega Dalton-sized ribosome pose special challenges due to the large size and complicated nature of the ribosome-nascent chain complex. This work introduces a combination of three-component analysis of fluorescence depolarization decays (including the presence of two local motions) and in-cone analysis of diffusive local dynamics to investigate the spatial constraints experienced by a protein emerging from the ribosomal tunnel. We focus on E. coli ribosomes and an all-alpha-helical nascent globin in the presence and absence of the cotranslationally active chaperones DnaK and trigger factor. The data provide insights on the dynamic nature and structural plasticity of ribosome-nascent chain complexes. We find that the sub-ns motions of the N-terminal fluorophore, reporting on the globin dynamics in the vicinity of the N terminus, are highly constrained both inside and outside the ribosomal tunnel, resulting in high-order parameters (>0.85) and small cone semiangles (<30 degrees ). The shorter globin chains buried inside the tunnel are less spatially constrained than those of a reference sequence from a natively unfolded protein, suggesting either that the two nascent chain sequences have a different secondary structure and therefore sample different regions of the tunnel or that the tunnel undergoes local structural adjustments to accommodate the globin sequence. Longer globins emerging out of the ribosomal tunnel are also found to have highly spatially constrained slow (ns) motions. There are no observable spectroscopic changes in the absence of bound chaperones.

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Figures

Figure 1
Figure 1
A: General features of the Lipari-Szabo model-free approach describing local motions of an axially symmetric probe (cylinder) by fluorescence depolarization techniques. B: Cartoon representations of the global and spatially confined local dynamics of a macromolecule in the presence of either one or two local motions. Both the fast (F) and intermediate (I) timescale local motions can be analyzed according to the in-cone-wobbling model, assuming all motions to be independent. The global macromolecular slow motions are denoted by the symbol S (not to be confused with the order parameter). C: General features of the in-cone wobbling model describing diffusive local dynamics. The vector (formula image), defining the fluorophore's symmetry axis, is colinear with either the emission or excitation dipoles of the fluorophore and is allowed stochastic wobbling motions (described by the angle θ) within a static cone defined by the cone semiangle θo, in a reference frame attached to the macromolecule with the z axis representing the normal to the macromolecular surface. The motions are randomly distributed in the XY plane and therefore independent of the azimuthal angle φ. The plot on the right illustrates the dependence of the order parameter S and its square S2 on the cone semiangle θo.
Figure 2
Figure 2
Fluorescence depolarization raw data, multicomponent fits, and curve fitting residuals for ribosome-bound and ribosome-released apoMb153.
Figure 3
Figure 3
A: Cone semiangles for the sub-ns motions of apoMb RNCs sensed by a BODIPY fluorophore covalently linked to the nascent chain N terminus. Data are shown for nascent chains of increasing length, up to the full-length 153-residue protein. RNCs were generated either from a wild type (gray bars) or a Δtig (dashed bars) cell strain. The resuspended RNCs generated from the Δtig strain were treated with the GrpE cochaperone and an excess of ATP to remove any DnaK chaperone bound to the nascent chains. The conditions are denoted as chaperone-free, in that the nascent chain contains no bound chaperone. Error bars comprise ± one standard error. B: Amplitude of the fast (sub-ns) motions experienced by the apoMb and reference PIR nascent polypeptides of increasing length under different conditions. Data were collected for samples prepared from either wild type (○, ▪) or Δtig trigger factor-depleted (×, ▵) cell strains. C: Cone semiangles for the intermediate timescale (ns) motions of apoMb RNCs. These motions are present only for the longest apoMb nascent chains. The 153-residue full-length protein is analyzed in the presence of ribosomes generated either from a wild type (squares) or TF-depleted Δtig (triangles) cell strain. In the latter case, nascent chains were also treated with GrpE and an excess of ATP (chaperone-free conditions).
Figure 4
Figure 4
Physical representations of the ribosomal exit tunnel and the shortest apoMb and PIR nascent chains examined in this work. All the ribosomal tunnel images were generated by rolling ball simulations starting from the Haloarcula marismortui structure, as detailed in Ref. 8. A: Space-filling side view of the ribosomal exit tunnel. The peptidyl transferase center (PTC) is in the back of the image. Lines 1–5 show the vertical tunnel slices used in panels D–G. These specific planes were selected because they yield representative views of the tunnel dimensions useful to evaluate the progression of the nascent chain inside the tunnel. B: Space-filling side-view of the ribosomal exit tunnel. C: Space-filling and chemical formula of the BODIPY-labeled nascent chain N terminus. D, F: Pictorial representations of the shortest apoMb and PIR nascent chains examined here, modeled as fully extended chains. The cones shown in the image have dimensions matching the experimental cone semiangles and consistent with the experimentally determined rotational correlation times. Different views showing distinct tunnel slices are displayed. E, G: Pictorial representations of the shortest apoMb and PIR nascent chains as in panels D and F, except that the nascent chains were modeled as α-helices rather than extended chains.
Figure 5
Figure 5
Variations in (A) cone semiangle θo and (B) apparent rotational correlation time before and after the release of full-length apoMb153 and PIR from the ribosome. Error bars denote ± one standard error.
Figure 6
Figure 6
A: Cartoon representation of the proposed equilibria among different types of apoMb153 RNC populations. TF denotes the trigger factor chaperone. Four species, labeled as 1–4, are postulated to exist. B: Graphical representation of the calculated percent populations of species 1–4 as a function of the binding affinity of TF for ribosome-bound nascent chains, expressed as the KD TF-RNC dissociation constant. This plot assumes that the binding affinity between TF and the ribosome is constant (according to published values38,40) and it does not depend on the affinity between nascent chains and TF. C: Results for low and high values of KD TF-RNC are reported. Supporting information includes the derivation of equations necessary for the plots in Figure 6 (panels B and C).
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