PMC

ΔG_H2O by fluorescence verses (a) ΔG_H2O by H/D kinetic-based method or (b) Δm {m by flurorescence- m by H/D kinetic-based method} for 14 staphylococcal nucleases. X-axis values taken from references ,,. Y-axis values derived from references (m by fluorescence) and this paper (m and ΔG_H2O by H/D kinetic-based method). Average uncertainty associated with ΔG_H2O is 0.3 kcal mol⁻¹ and for (Δm) is 0.1 kcal mol⁻¹ M⁻¹.

Average mass shift (ΔM) upon H/D exchange as a function of [GdHCl] for wild-type staphylococcal nuclease. Three curves corresponding to exchange times of 1.5 (○), 11 (□), and 19.5 (Δ) min. The average uncertainty associated with the mass shift estimations at different GdHCl concentrations is about ± 2 Da.

Staphylococcal nuclease wild type by PEPS method. The top panel (a) shows variations in peak intensities corresponding to the deuterated open and closed forms as a function of GdHCl concentration. The middle panel (b) shows Δ M (Δ) {right side y-axis}, the percent of folded protein (●) calculated using Δ M, and the percent of folded protein from fluorescence data (○) [,,]. All are plotted as a function of GdHCl concentration. The bottom panel (c) shows the ESI mass spectra which were deconvoluted to produce the data in panel (a).

Solvent denaturation plots using the linear extrapolation method (LEM) for three staphylococcal nucleases and ubiquitin obtained from HX ESI-MS kinetic-based method and PEPS HX ESI-MS method compared to literature fluorescence and CD data. For staphylococcal nuclease: open symbols represent the PEPS method and solid symbols represents the kinetic-based HX-MS methods; 23I/25I/66L/72L (Δ); wild type (□), 117G/124L/128A/41I/59A/21K/21N (○); dashed lines (1), (2) and (3) show fluorescence data [, ,]. For ubiquitin (◇) dashed line (4) represents literature NMR data [] and (▽) literature CD data [].

Average mass shift (ΔM) upon H/D exchange as a function of exchange time for ubiquitin. Seven curves correspond to different GdHCl concentrations: 0.00M, 0.48 M, 0.84 M and 1.26 M (▽), 1.56 M (■), 1.76 M (□), 1.92 M (▲) and2.16 M (○). The bottom (dotted) line corresponds to a closed state (minimum exchange); the middle (dashed) line corresponds to 50% exchange and the top (solid) line corresponds to the maximum exchange. In Figure 2b these lines have been adjusted to encompass only denaturation concentration dependent H/D exchange to facilitate assessment of 50% exchange in the denatured (unfolded) protein. The average uncertainty associated with the mass shift estimations at different GdHCl concentrations is about ± 2 Da