Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase

Abstract

We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale.

Figure 1. Purification and biophysical characterization of the MjPAN complex.

(A) Superose 6 column chromatography purification profiles of the dodecameric/hexameric mixture (blue chromatogram) and the hexameric (green chromatogram) forms of MjPAN complexes. Negative stain transmission electron micrographs of the MjPAN complex in its dodecameric (pool A) and hexameric (pool B) forms are shown next to the corresponding chromatogram. (B) Kinetic analysis of the interactions of PAN with GFPssrA. The colored lines represent binding responses for injections of protein analyte at specified concentrations (μM) over the PAN-coated surface. The kinetic data were fitted (black curves) by a 1:1 Langmuir binding model which describes monovalent analyte binding to a single site on the immobilized ligand (see SI Material and Methods).

Figure 2. GFPssrA is released and aggregates in solution after unfolding by PAN.

Middle: SANS curves recorded on deuterated (d) dGFPssrA (2 mg/ml) in the presence of hydrogenated (h) hPAN (10 mg/ml) and ATP (100 mM) in a 42% D₂O buffer (hPAN invisible) at 55 °C during the unfolding reaction. The back-calculated SANS curve (CRYSON35) from the GFP crystal structure (PDB ID 2B3P) is shown as a black continuous line. Top: ab initio envelopes of dGFPssrA at different times and the respective volumes of each model. The first model (grey) is generated from SANS data without hPAN (control experiment, see Fig. S3) and is superimposed with the crystal structure. Bottom left: pair distance distribution functions, P(r), calculated using GNOM.

Figure 3. Time-resolved quantification of native and aggregated GFPssrA populations during unfolding by PAN.

Curves showing the populations of natively folded GFPssrA (green) and its aggregates (red) during the unfolding reaction. Continuous lines display fits with Eq. 2. Inset: UV fluorescence spectroscopy emission spectra of GFP in the presence of PAN and ATP at 55 °C recorded on the same samples and at the same time as the SANS measurements. The GFP emission peak fluorescence at 509 nm is plotted in blue and was fitted by a single exponential decay function (Supplementary Eq. 4).

Figure 4. ATP-induced reversible contraction of PAN during substrate unfolding.

Middle: Scattering curves recorded on deuterated (d) dPAN (3 mg/ml) in the presence of hydrogenated (h) hGFPssrA (4.8 mg/ml) (invisible in 42% D₂O buffer) at 60 °C with 30 s exposure time per curve during different times of the unfolding process. Inset, left and bottom: Guinier fits and pair distance distribution functions, P(r), calculated using GNOM. Inset, top: schematic representation of a putative conformational transition from a relaxed to a contracted PAN state upon ATP hydrolysis involving N-ter coiled coil movements and C-ter ATPase domain conformational changes, rotations and translational movements.

Figure 5. Time-dependence of the contraction and re-expansion of PAN during substrate unfolding.

Evolution of the (ensemble-averaged) PAN radius of gyration as a function of reaction time. PAN is shown schematically in a contracted and extended state. A two exponential function (Eq. 3) is fitted against the experimental data (black curve). Inset: UV fluorescence spectroscopy emission spectrums of GFPssrA in the presence of PAN and ATP at 60 °C recorded on the same samples and at the same time as the SANS measurements. A single, typical experimental error bar is shown for the R_G data at 40 min.

Figure 6. Putative scheme representing GFP unfolding by PAN.

(A) At the beginning of the reaction PAN is in a relaxed, extended state and all GFP molecules are in their native state with maximum fluorescence. (B) After a few minutes PAN reaches its maximum activity and destabilizes/unfolds GFP molecules. GFP molecules are either released in a destabilized, yet fluorescent state or in a completely denatured, unfluorescent state and form first, small aggregates. (C) At a later state all natively folded GFP molecules have disappeared and all destabilized or denatured GFP molecules are clustered within aggregates. Occasionally, PAN interacts with clusters and denatures the destabilized members.