Skip to main content
. 2022 Aug 29;41(23):e111344. doi: 10.15252/embj.2022111344

Figure EV2. Refolding kinetics analysed with global HDX‐MS of PpiA and PpiB at 25 and 4°C (related to Fig 2).

Figure EV2

  • A
    Pipeline of processing in vitro refolding kinetics of intact proteins using global HDX‐MS analysis and subsequent visualization as a colour map (Fig 2B). (i) Denatured proteins are refolded out of chaotrope (6 M urea) into aqueous buffer where the different folding states are observed. (ii) An aliquot of the refolding reaction is removed at different timepoints and pulse‐labelled in high % D2O where the amount of Deuterium taken up reflects the number of non‐H‐bonded/solvent‐accessible backbone amides and is inversely related to how folded (i.e. stably H‐bonded) the protein is. The unfolded state (6 M Urea) is experimentally defined as a single peak/population with maximum D‐uptake (set as 100%), followed by intermediate D‐uptake and finally the lowest D‐uptake for the folded state. (iii) From the electrospray ionisation MS analysis of each refolding timepoint, an m/z spectrum with multiple charged m/z peaks is obtained. From the latter, a single high‐intensity peak (highest Signal over Noise) is selected and smoothed (Savitzky‐Golay, window: 15, number: 5) to be followed over different refolding timepoints (as depicted in the bottom section). Due to Deuterium being 1 Da heavier than Hydrogen, a shift from a high to lower m/z is observed over time as the protein folds and takes up fewer Deuterium during pulse‐labelling. “o”: Potassium adducts and Urea modification peaks that are visible on the (un)folded state. (iv) The intensities of the folding populations from the single m/z peak at different timepoints are normalized to the integrated area (See Materials and Methods). To observe the conversion of the folding populations over time, a 3D plot was displayed with all the normalized m/z spectra over time. The normalized intensities now reflect the population fractions of each folding state. Linear interpolation was performed between the m/z spectra over time to get a continuous time course of the refolding pathway and used to create a 2D colour map to visualize the interconversion between folding states indicated based on their degree of unfoldedness (%D‐uptake). The colour gradient (“magma” colourmap) reflects increasing population fractions ranging from small (dark) to high (yellow; see Materials and Methods, Dataset EV3C).
  • B, C
    Smoothed spectra of the 24+ charged m/z peak (highest intensity) of the refolding kinetics of PpiA and PpiB from global HDX‐MS analysis at the indicated timepoints (4 and 25°C) that were used for constructing the continuous colour map (Fig 2B). The denatured protein or the fully deuterated (FD; 6 M Urea‐d4 for 1 h; red line) control and the Native control (i.e. soluble purified native protein; blue line) are marked throughout the folding timepoints. “o” refers to Potassium adducts and Urea modification peaks that are also visible in the colour maps in Fig 2B.
  • D
    Population fraction over time after Lorentzian curve fitting of the 24+ charged m/z peak in (B and C) with the unfolded (red), intermediate (purple) and folded (blue) state (from biological repeats, see below). The relative percentage of D‐uptake of each intermediate state is noted in its subscript. For PpiB at 25°C (n = 2), PpiA at 25°C (n = 2), PpiB at 4°C (n = 7) and PpiA (n = 4), data are shown as dots (up to 3 repeats) and average as line.
  • E
    For 4°C, the population fractions were fitted with an ODE model (see equation, see Materials and Methods). The fitted curves are displayed with the different folding states (Unfolded (U), Intermediate (I) and Folded (F)) and the equilibrium constant K1 is displayed below. n = 4 biological repeats.
  • F
    Refolding of PpiA (orange) and PpiB (green) monitored by CD at 4°C (recorded at 222 nm and shown as the folded fraction over time setting the 6 M Urea state as 0 and the final 0.2 M Urea state as 1).