Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations

Moritz Mickler, Ruxandra I. Dima, Hendrik Dietz, Changbong Hyeon, D. Thirumalai, and Matthias Rief
PNAS December 18, 2007 104 (51) 20268-20273; https://doi.org/10.1073/pnas.0705458104

  1. Edited by José N. Onuchic, University of California at San Diego, La Jolla, CA, and approved October 25, 2007 (received for review June 11, 2007)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    Time-dependent changes in the force and root-mean-square deviations [ΔΩ(t)]. (a) Changes in the force for cycle 3 GFP for molecules that reach state by the major pathway. (Upper) The structures of the three metastable intermediates along the unfolding pathway are shown and the secondary structural elements that detach are labeled. (Lower) ΔΩ(t) is shown for different structural elements Ω (Ω for black, red, blue, and green correspond to WT, α, αβ1, α[β1 − β3], respectively). (b) Same as a, except the forces and the structures are for molecules that unfold along the minor pathway. (Lower) ΔΩ(t) is shown for various fragments. The green curve corresponds to Δαβ11(t).

  • Fig. 2.
    Fig. 2.

    Mechanical unfolding of wild-type GFPΔα at two temperatures. (a) Typical unfolding pattern of GFPΔα at 20°C. Solid lines show worm-like chain fits to the data. (b and d) Histogram of the length gains observed for partial unfolding of GFPΔα into subsequent intermediate states (see arrows in a and c). (c) Typical unfolding pattern of GFPΔα at 8°C.

  • Fig. 3.
    Fig. 3.

    Internal cysteine-engineering to alter the unfolding pathway of GFPΔα. (a) Scheme of GFPβC-lock. (b) Histogram of the length gain on unfolding of single GFPβC-lock molecules from GFPΔα to the fully unfolded state. Green section indicates the population where the disulfide bond has been successfully formed. (c) Overlay of several experimental force-extension traces for unfolding of GFPβC-lock, with the disulfide bond formed. The population of intermediate states, manifested by dwell levels in the cantilever relaxation phase (gray-shaded zone), is clearly visible. (d) Scheme of GFPβN-lock. (e) Histogram of the length gain on unfolding of single GFPβN-lock molecules from GFPΔα to the fully unfolded state. Red section indicates the population where the disulfide bond has been successfully formed. (f) Overlay of several experimental force-extension traces for unfolding of GFPβN-lock, with the disulfide bond formed. Dwell levels in the cantilever relaxation phase (gray-shaded zone) are consistently absent. (g) Scheme of GFPβN-lock under reducing conditions. (h) Histogram of the length gain on unfolding of single GFPβN-lock molecules from GFPΔα to the fully unfolded state as observed in experiments under reducing buffer conditions (20 mM DTT). Red section indicates the population where the disulfide bonds persist even under the action of DTT. (i) Overlay of several experimental force-extension traces for unfolding of reduced GFPβN-lock. Dwell levels in the cantilever relaxation phase are again consistently observed.

  • Fig. 4.
    Fig. 4.

    Histogram of the forces required to induce unfolding of wild-type GFPΔα (black line), GFPβC-lock (red line), and GFPβN-lock (green line).

  • Fig. 5.
    Fig. 5.

    Simulation results for force-extension curves and ΔΩ(t) for cross-link mutants. (a) Force as a function of extension for GFPβN-lock mutant. Residues 11 and 36 are connected by a disulfide cross-link. The structural changes that accompany the unfolding process are shown by blue arrows. The bottom profile gives ΔΩ(t) as a function of t for Ω = WT (black), Ω = α (red), and Ω = αβ11 (green). (b) Same as a, except the FEC and ΔΩ(t) are for the GFPβC-lock mutant in which residues 202 and 225 are covalently linked by a disulfide bond. In addition to the ΔΩ(t) shown in a, the purple gives Δα[β1 − β3](t).

  • Fig. 6.
    Fig. 6.

    The complex energy landscape for GFP unfolding is constructed based on the results of simulations and experiments. Starting from the folded structure, which corresponds to the native basin of attraction, unfolding occurs by bifurcation in the pathway after the rupture of the α-helix. The molecules that unfold by the dominant pathway are shown by green arrows and the purple arrows show the fate of GFP molecules that follow the minor pathway. The structures of the intermediates in the various basins are explicitly shown. The approximate fraction of the molecules along each pathway is indicated. These numbers can be altered by mutations (cross-link in this study) and by changing the force direction. Thus, the energy landscape is not only rugged, but also can be manipulated.

Tables

  • Table 1.

    Frequency of observing dwell levels (detection limit ≥ 100 μs) in the cantilever relaxation after GFPΔα unfolding, reflecting population of the GFPΔαΔβ intermediate state

    Intermediate, %No intermediate, %
    Wild-type GFP40.859.2
    GFPβC-lock 72.727.3
    GFPβN-lock 0100

Data supplements

  • Mickler et al. 10.1073/pnas.0705458104.

    Supporting Information

    Files in this Data Supplement:

    SI Text
    SI Figure 7
    SI Figure 8
    SI Figure 9
    SI Table 2




    Fig. 7. (Upper) Time-dependent changes (obtained by using simulations) between various strands in cycle 3 GFP for one trajectory in which unfolding begins with the rupture of the C-terminal b-strand. Such a process represents the minor pathway in the mechanical unfolding of cycle 3 GFP. (Lower) Changes in the angles between the strands. The color schemes in Upper and Lower are identical. The complete rupture of strands leading to an increase in d also coincides with the changes in the corresponding angle (see, for example, the green and blue curves).





    Fig. 8. Fluorescence spectra of cycle 3 GFP (black) compared to GFPbC-lock (green) and GFPbN-lock (red). (a) Emission was detected at 508 nm. (b) The fluorescence was excited at 400 nm. Both emission and excitation spectra of the cross-link mutants are identical, indicating that the structure of GFP is unaltered by cysteine mutations.





    Fig. 9. Same as Fig. 7 except the plots are for GFPbN-lock. The changes in d and f for the mutant are drastically different from cycle 3 GFP (see Fig. 7). The analysis of these curves indicates that for the GFPbN-lock there is only one populated intermediate, namely, GFPDa (see main article for additional discussion).

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Revealing the bifurcation in the unfolding pathways of GFP by using single-molecule experiments and simulations
Moritz Mickler, Ruxandra I. Dima, Hendrik Dietz, Changbong Hyeon, D. Thirumalai, Matthias Rief
Proceedings of the National Academy of Sciences Dec 2007, 104 (51) 20268-20273; DOI: 10.1073/pnas.0705458104
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