Action of the chaperonin GroEL/ES on a non-native substrate observed with single-molecule FRET

Abstract

The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.

Figure 1

(a) The crystal structure of VHL in the VBC complex and the locations of Cys mutations and the inter-residue distances in this study. Hydrophobic binding sites are highlighted in red (Box1 or B1) and blue (Box2 or B2). (b) The table shows the sequence separation in number of amino acids and the distance between two positions in the VBC complex for the various double-Cys variants. (c) Bulk FRET efficiencies estimated by the proximity ratio F for 6 VHL variants in 6M GdnHCl, in native buffer, and with GroEL. The double-labeling efficiency was ~95%, as HPLC purification allowed separation of the doubly-labeled fraction from singly-labeled and unlabeled. For the native buffer case, denatured VHL in 6M GdnHCl was diluted into buffer A (50 mM HEPES-KOH (pH 7.4), 50 mM KCl, 10 mM MgCl2, 10% glycerol, 1mM DTT), with a 1:100 volume ratio (100 nM final concentration). To confirm that VHL was not aggregated in this condition, we separately used donor-only labeled and acceptor-only labeled VHL, diluted into the native buffer with the same final concentration, and found no significant FRET, indicating that VHL is not aggregated in this condition. In the case of VHL with GroEL, we removed any unbound VHL by treating the solution with a spin column after incubation. This causes a concentration variation of each sample, and therefore the proximity ratio is reported as a measure of relative FRET efficiency.

Figure 2

(a) PEG-coated glass coverslips were prepared as described by Joo, et al. (Bottom) A specific binding test was performed using biotin- and Cy3-labeled GroEL (C473Bio-EL). In the presence of Neutravidin (left panel), Cy3-labeled C473Bio-EL could be viewed on the surface, producing more than ~30 fluorescence spots in the viewing region (~10×10 μm). On the other hand (right panel), as a negative control, after washing any unbound C473Bio-EL, only 1–2 fluorescence spots could be observed in the absence of Neutravidin. (b) Single-molecule polarization histograms of the AX488 dye spun on a glass coverslip (left), AX488 labeled VHL (N)-GroEL (middle), TR labeled VHL (N)-GroEL (right). For the latter two cases, single VHL-GroEL complexes were immobilized on the PEG-coated glass coverslip using the biotin-Neutravidin linkage. The narrow distribution centered at P=0 for the VHL-GroEL complexes implies that the GroEL-VHL complexes are free to rotate without any specific interaction with the surface on the 100 ms time scale.

Figure 3

Single-molecule FRET efficiency distributions measured with individual VHL-GroEL molecules. After preparation of the biotin-PEG coated surface, complexes were prepared by diluting unfolded VHL into buffer A with C473Biotin-EL for 20 min, incubated on the surface for 5 min, and the surface was then washed before imaging. To produce the lid closed state, the VHL-C473Biotin-EL complex was further incubated with ATP/AlFx for 30 min, and then with GroES for 10 min. Single molecules were imaged with 488 nm pumping in two color detection channels to record both the emission from the donor and from the acceptor simultaneously. Illumination was performed in the time-lapse mode for 100 ms followed by a dark period of 10 s; under these conditions single molecules survived for time periods on the order of 10 s total elapsed time (see Fig. 4). Four VHL variants labeled at two positions (N-B1, B1-B2, B2-C and N-C) are shown in this figure. The upper row shows the case for the lid-open state, i.e., GroEL-VHL complexes alone. Dotted lines in each graph indicate the average FRET efficiency for the lid-open state. The lower row shows the case for the lid-closed state produced by the addition of GroES and ATP/AlFx.

Figure 4

(a) The number of surviving molecules distribution for single molecules of AX488 VHL (N) with GroEL. Continuous illumination with 0.1 sec integration time was used to measure the number of molecules remaining after specific illumination times to assess photobleaching, while time-lapse imaging (0.1 sec exposure with 0.9 sec dark interval, inset) was used for longer time observations. The continuous illumination distribution was fit with a single exponential, with the time constant τ = 4.1 sec. The inset shows the number of surviving molecules under time-lapse conditions with abscissa showing in real laboratory elapsed time with ATP addition in the presence of GroES. ATP was added after 15 cycles of time-lapse (dotted line), which corresponded to t = 1.5 sec of actual illumination. The number of molecules visible on the surface significantly dropped right after ATP addition, due to the disappearance of some VHL molecules caused by release of some VHL molecules into the solution. (b) Examples of time-lapse single-molecule fluorescence traces of donor/acceptor emission and calculated FRET efficiency values for GroEL-bound VHL before/after ATP addition with GroES present in the solution. This is a complex where VHL did not immediately dissociate after ATP addition. Arrows indicate when ATP was added (t = 15 sec). The example on the left (a B1-B2 variant) showed increased FRET after ATP addition and photobleaching at 40 sec, whereas FRET decreased in the N-C example on the right with photobleaching at 37 sec.

Figure 5

Characterization of individual FRET transitions of (a) single B1-B2 and (b) single N-C molecules upon ATP addition. The calculated distances extracted from true FRET efficiency E before and after ATP addition were used as x- and y- coordinates. Only transitions with significant changes before/after ATP addition are shown. The solid lines represent no change after ATP addition. In the case of the B1-B2 variant, 84% of molecules showed compaction (distance-decrease transition) after ATP addition, while 69% of N-C variant molecules showed expansion (distance-increase transition). (c) Summary of the structural changes of VHL upon binding to GroEL followed by cavity closure (only one ring shown). First, unfolded VHL shows compaction upon binding to GroEL. Further structural changes could be observed after ATP-induced lid closure, but these were quite heterogeneous with net average compaction for B1-B2 and net expansion for N-C.