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. 2016 Nov 22;113(47):13402-13407.
doi: 10.1073/pnas.1610272113. Epub 2016 Nov 7.

Quantitative determination of ribosome nascent chain stability

Avi J Samelson  1   2 , Madeleine K Jensen  1   2 , Randy A Soto  2 , Jamie H D Cate  1   2   3 , Susan Marqusee  4   2
Affiliations

Quantitative determination of ribosome nascent chain stability

Avi J Samelson et al. Proc Natl Acad Sci U S A. .

Abstract

Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein's energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.

Keywords: cotranslational folding; protein folding; protein stability; pulse proteolysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Comparison of spectroscopy and pulse proteolysis for determining protein stability. Samples are equilibrated in urea overnight for both spectroscopic methods (Left) and pulse proteolysis (Right). For spectroscopic methods, equilibrated samples are read directly in a spectrophotometer to determine the signal difference between the folded and unfolded states. Alternatively, for pulse proteolysis, a protease is added to digest all unfolded protein at a given urea concentration. Samples are analyzed by SDS/PAGE, and the remaining band represents the amount of folded protein at each urea concentration. The intensity of each band is plotted against the urea concentration and then fit to determine the concentration of urea where half of the folded protein remains, or the Cm. The Cm is multiplied by the protein’s m-value to determine the stability, ∆Gunfolding.
Fig. 2.
Fig. 2.
Stability of proteins purified from E. coli and made using IVT by pulse proteolysis. (A) DHFR V75R purified (in gray and upper gel) and in vitro translated (in blue and lower gel). (B) RNase H I53D purified (in gray and upper gel) and in vitro translated (in green and lower gel). Note that the concentration of urea is different for each lane between gels. Each gel and trace shown is representative of three separate experiments. Error bars represent the SD of the Cm for each curve plotted, determined by three separate experiments.
Fig. S1.
Fig. S1.
Methotrexate (MTX) binds in vitro translated DHFR V75R. (A) Gray indicates the fraction folded as a function of urea for in vitro translated DHFR V75R. Blue indicates the fraction folded as a function of urea for in vitro translated DHFR V75R in the presence of 2 μM MTX. (B) Gel of MTX used for quantitation in A.
Fig. 3.
Fig. 3.
Urea sensitivity of 70S ribosomes and RNCs. (A and B) Sucrose gradient ultracentrifugation of 70S ribosomes (A) and RNCs (B). Highlighted in blue is the peak corresponding to 70S ribosomes. The 50S peak is highlighted in green; the 30S peak, in yellow. The 70S ribosomes and RNCs were also run in gradients containing no magnesium and 0.5 M KCl as a negative control. (C) Diffusion coefficients of RNCs plotted as a function of urea concentration as determined by FCS. Error bars represent the SD of at least 10 experiments. Fits are shown in Fig. S5.
Fig. S5.
Fig. S5.
Sample FCS runs with fits for each urea concentration. Colors represent four different acquisitions. Diffusion coefficients of RNCs are plotted as a function of urea concentration. Error bars represent the SD of at least 10 experiments.
Fig. 4.
Fig. 4.
Determination of RNC stability by pulse proteolysis. (A) DHFR V75R RNCs. Blue indicates protein on the ribosome; gray, protein off the ribosome. (B) RNase H I53D RNCs. Green indicates on the ribosome; gray, off the ribosome. (C) Barnase W35F/W94F/H102M RNCs. Purple indicates on the ribosome; gray, off the ribosome. Each gel and trace shown is representative of three separate experiments. *Incomplete tRNA digestion. #RNase A. Error bars represent the SD of the Cm for each curve determined by three separate experiments, except for Barnase, for which the Cm is the average of two experiments.
Fig. S2.
Fig. S2.
The presence of the ribosome does not inhibit protease accessibility. (A) TEV cleavage of DHFR V75R-TEV-(GS)5-SecM as a function of time after RNase A digestion overnight. (B) Quantification of gel band intensities normalized to the highest band intensity on the gel. The original band is shown as filled circles; the band corresponding to cleavage product, as open circles. (C and D) Same as A and B, respectively, but as RNCs. Each gel and point shown is representative of three separate experiments. Error bars represent the SD of the normalized band intensity for each time point.
Fig. 5.
Fig. 5.
Folding on the ribosome is reversible. DHFR V75R RNCs reached equilibrium in either 0.5 M or 2.5 M urea. Samples were then split in half and diluted to 0.5 M urea or 2.5 M urea. (A) After another equilibration step, each sample was again split in half and either treated or not treated with thermolysin to assess the amount of folded protein remaining, and then run on a gel. Starred bands are due to incomplete RNase A digestion of attached tRNA. (B) Gel showing complete cleavage of peptidyl-tRNA as a function of time. (C) Quantitation of the data shown in A. Because there is the same amount of folded protein independent of the initial concentration of urea, folding is reversible. Error bars represent the SD of duplicate experiments.
Fig. 6.
Fig. 6.
RNC stability increases as the distance to the PTC increases, as determined by pulse proteolysis. (A) DHFR V75R RNC stability as a function of linker length. Blue, 35 aa from PTC; purple, 45 aa; red, 55 aa. (B) Same as A but with a stalling-deficient SecM mutant, 1FSTPVWISQAQGIAAGA17. (C) RNase H I53D RNC stability as a function of linker length. Green, 35 aa from PTC; orange, 45 aa; yellow 55 aa. (D) Same as C except after RNase A digestion overnight. Each trace shown is representative of three separate experiments. Error bars represent the SD of the Cm determined by three separate experiments. (E) Constructs used in this study. The sequence coding for each target protein was appended with a variable size glycine-serine linker, (GS)x, followed by the SecM stalling sequence at its C terminus. Gels of traces plotted here are present in Fig. S6.
Fig. S6.
Fig. S6.
Gels used in Fig. 6. (A) DHFR V75R-(GS)x-SecM on ribosome gels. (B) DHFR V75R-(GS)x-SecM off ribosome gels. (C) RNase H I53D-(GS)x-SecM on ribosome gels. (D) RNase H I53D-(GS)x-SecM off ribosome gels.
Fig. S3.
Fig. S3.
Purification of labeled RNCs. IVT reactions (input) were loaded onto a sucrose cushion and centrifuged as described in Materials and Methods. Supernatant (sup) was aspirated, and the pellets were washed three times (W1, W2, and W3) with 200 μL of ice-cold HKM+DTT. Pellets were then resuspended, mixed with SDS/PAGE loading dye, and loaded on a gel.
Fig. S4.
Fig. S4.
Diffusion of Alexa Fluor 488 as a function of urea concentration. The diffusion of Alexa Fluor 488 was used to determine the viscosity of the solution when calculating diffusion coefficients of RNCs. Shown here is the apparent diffusion coefficient without correcting for changes in viscosity due to the presence of high concentrations of urea. Error bars are SDs of 15 experiments.
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