Research ArticleCELL BIOLOGY

Kinetic and structural comparison of a protein’s cotranslational folding and refolding pathways

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Science Advances  30 May 2018:
Vol. 4, no. 5, eaas9098
DOI: 10.1126/sciadv.aas9098
  • Fig. 1 Cotranslational folding of HaloTag monitored in real-time with FP.

    (A) Cartoon representation of the crystal structure of HaloTag from PDB 5UY1 (41). Cys61 and Cys262, as well as the active-site Asp (D106), are represented as spheres. (B) Secondary structure map of HaloTag (42). (C) Refolding of HaloTag as a function of time monitored by FP. HaloTag refolding as a function of denaturant concentration (inset). (D) HaloTag cotranslational folding (blue; left axis) measured by FP and HaloTag synthesis (black circles with red fit; right axis) measured by gel (see fig. S1). Elongation rate as a function of time is shown (inset). aa, amino acid.

  • Fig. 2 Characterization of HaloTag folding kinetics and stability.

    (A) Chevron plot of HaloTag folding and unfolding rates as a function of urea concentration. Fast phase (black circles) and slow phase (white circles, black outline). Refolding as measured by FP is shown in blue. Refolding traces of HaloTag at (B) 0.8 M urea, where there is visible protein aggregation, and (C) 1.6 M urea, where no precipitation is observed. (D) CD spectrum of HaloTag at 0 M urea. (E) Equilibrium denaturant melt of HaloTag. (F) Burst-phase amplitudes for refolding (white triangles with black outline) and unfolding (white squares with black outline). Kinetic final amplitudes (black circles) overlay well with the fit of equilibrium data (blue line). Error bars represent the SD of three separate experiments.

  • Fig. 3 HaloTag folding is more efficient during in vitro translation than after refolding.

    (A) Fraction of total protein remaining in supernatant after centrifugation following refolding of HaloTag to 0.8 M urea. (B) Fraction folded as measured by pulse proteolysis in conditions as indicated—either after refolding, after in vitro translation, or both. Blue circles are in vitro–translated protein. (C) Representative gels for (A) and (B). All error bars are the SDs of at least 15 separate experiments except for HaloTag in 0.8 and 8.0 M urea, which are the SDs of three experiments. *P < 0.01, Student’s unpaired t test.

  • Fig. 4 The HaloTag folding trajectory changes during cotranslational folding.

    (A) Peptides derived from HX-MS experiments after 10 s of refolding were plotted according to their corresponding secondary structural element. Helices are lettered, whereas β sheets are numbered. Secondary structural elements were then divided into fast-folding (red circles) or slow-folding (blue circles) regions based on the average fraction deuterated (solid line) for peptides within those secondary structures at the 10-s time point (above or below dashed line). Error bars represent SEM. (B) Normalized fraction deuterated for all peptides (filled circles) plotted with the mean fraction deuterated for each group of peptides (solid lines) is shown for three time points. A full list of peptides is available in table S4. (C) Crystal structure of HaloTag with slow (blue) and fast (red) secondary structural elements colored. Loops are colored in white. Cysteines probed in (D) to (G) are represented as spheres (yellow, Cys61 and Cys262; purple, M129C; blue, I126C; green, E121C). (D to G) Cysteine accessibility experiments during in vitro translation (colored lines and circles) and refolding (dotted lines and black dots). (D) WT HaloTag. (E) Halo* M129C. (F) Halo* I126C. (G) Halo* E121C. Error bars represent the SD of three separate experiments except for (A) where error bars are the SEM. Gels are shown in fig. S6. AU, arbitrary units.

  • Table 1 Summary of kinetic and thermodynamic data of HaloTag and its mutants (error bars are SDs of at least three separate measurements).
    Data from equilibrium experiments
    ΔGunf (kcal⋅mol−1) (CD)6.03 ± 0.39
    m value (kcal⋅mol−1 M−1)1.57 ± 0.11
    Data from kinetic experiments
    ΔGunf (kcal⋅mol−1)5.24 ± 2.0
    m value (kcal⋅mol−1 M−1)1.41 ± 0.58
    kf,H2O (s−1)0.04 ± 0.02
    mf (kcal⋅mol−1 M−1)1.46 ± 0.71
    kconstant, H2O (s−1)6.6 ± 0.71 × 10−4
    mconstant (kcal⋅mol−1 M−1)0.02 ± 0.1
    kf, H2O,FP (s−1)4.8 ± 0.6 × 10−4
    mFP (kcal⋅mol−1 M−1)−0.1 ± 0.04
    kNI, H2O (s−1)8.47 ± 20 ± 10−6
    mNI (kcal⋅mol−1 M−1)−0.44 ± 0.3
    kIU, H2O (s−1)3.3 ± 9.9 × 10−4
    mIU (kcal⋅mol−1 M−1)0.70 ± 0.15
    Data from cysteine accessibility experiments
    kWT,refolding, slow (s−1)7.8 ± 0.6 ± 10−4
    kWT,refolding, fast (s−1)0.03 ± 0.02
    kWT,IVT (s−1)4.7 ± 0.3 × 10−4
    kM129C,refolding (s−1)>0.01
    kM129C,IVT (s−1)3.2 ± 0.2 × 10−4
    kI126C,refolding (s−1)>0.01
    kI126C,IVT (s−1)2.2 ± 0.2 × 10−4
    kE121C,refolding (s−1)>0.01
    kE121C,IVT (s−1)>0.01

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaas9098/DC1

    fig. S1. Cotranslational folding of HaloTag can be measured using FP.

    fig. S2. Addition of the peptidyl-proline isomerase CypA does not affect HaloTag refolding or cotranslational folding rates.

    fig. S3. Aggregation of HaloTag.

    fig. S4. Cysteine accessibility of WT HaloTag.

    fig. S5. Characterization of Halo* cysteine mutants.

    fig. S6. Gels for data shown in Fig. 4.

    fig. S7. Characterization of Halo* E121C cysteine accessibility.

    table S1. Kinetic data obtained for HaloTag folding using FP.

    table S2. Determination of HaloTag folding efficiency under different conditions.

    table S3. Folding rates of HaloTag and variants measured by FP.

    table S4. Normalized HX-MS data.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Cotranslational folding of HaloTag can be measured using FP.
    • fig. S2. Addition of the peptidyl-proline isomerase CypA does not affect HaloTag refolding or cotranslational folding rates.
    • fig. S3. Aggregation of HaloTag.
    • fig. S4. Cysteine accessibility of WT HaloTag.
    • fig. S5. Characterization of Halo* cysteine mutants.
    • fig. S6. Gels for data shown in Fig. 4.
    • fig. S7. Characterization of Halo* E121C cysteine accessibility.
    • table S1. Kinetic data obtained for HaloTag folding using FP.
    • table S2. Determination of HaloTag folding efficiency under different conditions.
    • table S3. Folding rates of HaloTag and variants measured by FP.
    • Legend for table S4

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    Other Supplementary Material for this manuscript includes the following:

    • table S4 (Microsoft Excel format). Normalized HX-MS data.

    Files in this Data Supplement:

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