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. 2018 Oct 2;115(40):E9280-E9287.
doi: 10.1073/pnas.1812756115. Epub 2018 Sep 17.

Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

José Arcadio Farías-Rico  1 , Frida Ruud Selin  1 , Ioanna Myronidi  1 , Marie Frühauf  1 , Gunnar von Heijne  2   3
Affiliations

Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome

José Arcadio Farías-Rico et al. Proc Natl Acad Sci U S A. .

Abstract

During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of the Escherichia coli ribosomal protein S6 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

Keywords: arrest peptide; protein folding; pulse proteolysis; ribosome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) Schematic representation of in vitro translated constructs. The protein under study is placed L residues upstream of the last proline in the AP, and a C-terminal 23-residue extension is added downstream of the AP to ensure that the arrested and full-length forms of the construct can be cleanly separated by SDS/PAGE. (B) The AP-based assay: At linker length L1, the protein is too deep in the exit tunnel to be able to fold, and hence no force is generated on the AP and mostly arrested nascent chains are produced (fFL ≈ 0). At L2, if the linker is stretched beyond its equilibrium length the protein can just reach a location in the exit tunnel where there is sufficient space for it to fold. Some of the folding free energy is therefore stored as elastic energy in the linker, increasing the force on the AP. More full-length protein is produced (fFL > 0). At L3, finally, the protein is already folded when the ribosome reaches the C-terminal end of the AP, and little force is exerted on the AP (fFL ≈ 0). (C) The on-ribosome pulse proteolysis assay: At L1, the protein is located too deep in the exit tunnel to be able to fold and the nascent chain is hence degraded by a brief thermolysin pulse. At L2, the protein is folded and hence resistant to proteolysis. Note that the protein is attached to the ribosome via a (GS)n linker that in itself is insensitive to thermolysin.
Fig. 2.
Fig. 2.
Fraction full-length protein (fFL) and thermolysin-resistant protein (fTR) plotted as function of L for the eight proteins discussed in the text. Each data point is an average of three independent experiments (error bars indicate SEM values). Lonset is the L value corresponding to half-maximal peak height (indicated by dotted lines), and Lmax is the L value corresponding to the maximum of the peak. (A) FSD1. The red data point at L = 17 residues is for the destabilizing mutation F21P. The blue data point at L = 61 residues (arrow) is for a construct where the (GS)n linker was replaced by a linker derived from the LepB protein (Materials and Methods). (B) WW domain. The red data point at L = 25 residues is for the destabilizing mutation Y19A. (C) Designed protein EHEE_rd2_0005. (D) Protein G, the red data point at L = 30 residues is for the destabilizing mutation F52L. (E) Calmodulin EF-2–EF-3. The black profile was obtained in 3 mM CaCl2, and the red profile in 0.3 mM EGTA. (F) Ribosomal protein S6. The black profile was obtained with the SecM(Ec) AP, and the gray profile with the SecM(Ms) AP. (G) SOD1 (lacking the metal-loading loops IV and VII). The full curve (squares) is the fFL profile and the dashed curve (triangles) is the thermolysin-resistance profile (fTR). (H) ILBP. The full curve (squares) is the fFL profile and the dashed curve (triangles) is the thermolysin-resistance profile (fTR).
Fig. 3.
Fig. 3.
Correlation between fFL and ΔGD-N values for wild-type S6 and 16 point mutations listed in SI Appendix, Table S3. The best-fit linear regression line is shown (fFL = 0.15 ΔGD-N − 0.41; R2 = 0.70; P = 2 × 10−5). Statistical analysis was performed using the StatPlus:mac Pro software.
Fig. 4.
Fig. 4.
(A) NMR structure of wild-type S6 from T. thermophilus (PDB ID code 2kjv). Positively charged residues (Arg+Lys) are colored in blue and negatively charged residues (Asp+Glu) in red. (B) fFL profile of wild-type S6+16–16 (for sequences see SI Appendix, Fig. S2). (C) fFL profile of S60. (D) fFL profile of S6+16–9. (E) fFL profile of S6+9–16. (F) fFL profile of S6−16. (G) fFL (squares, solid curve) and fTR (triangles, dashed curve) profiles for wild-type S6. Red data points at L = 17 and 57 residues are for the destabilizing mutation L10A. (H) fFL (squares, solid curve) and fTR (triangles, dashed curve) profiles for S6−16. Red data points at L = 17 and 57 residues are for the destabilizing mutation L10A. The SecM(Ec) AP was used in A–F and the SecM(Ms) AP in G and H.
Fig. 5.
Fig. 5.
Plot of Lonset vs. protein size for proteins included in this and previous studies. The shaded gray area indicates the approximate location of the distal end of the exit tunnel, and numbers on the right indicate the approximate distance from the peptidyl transferase center. Error bars of ±2.5 residues have been added to indicate that the fFL profiles in Fig. 2 were recorded with a five-residue resolution, hence introducing an uncertainty in the Lonset values. The dotted curve shows the linear best-fit obtained from a log-log plot: log10(Lonset) = 0.42 (±0.05) ∙ log10(size) + 0.69 (±0.1) (R2 = 0.80; P = 1.4 × 10−6). Proteins are indicated as follows: 1, FSD1; 2, ADR1a1; 3, WW domain; 4, EHEE; 5, protein G; 6, calmodulin EF-2–EF-3; 7, titin I27; 8, S6; 9, TOP7; 10, spectrin β16; 11, spectrin R16; 12, PENT; 13, spectrin R15; 14, SOD; 15, FLN5; 16, ILBP; and 17, DHFR (see SI Appendix, Table S5 for references).
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