Structure Characterization of a Disordered Peptide Using In-Droplet Hydrogen/Deuterium Exchange Mass Spectrometry and Molecular Dynamics

Abstract

In-droplet hydrogen/deuterium exchange (HDX)-mass spectrometry (MS) experiments have been conducted for peptides of highly varied conformational type. A new model is presented that combines the use of protection factors (PF) from molecular dynamics (MD) simulations with intrinsic HDX rates (k _int) to obtain a structure-to-reactivity calibration curve. Using the model, the relationship of peptide structural flexibility and HDX reactivity for different peptides is elucidated. Additionally, the model is used to describe the degree of conformational flexibility and structural bias for the disease-relevant Nt17 peptide; although highly flexible, intrinsically primed for facile conversion to α-helical conformation upon binding with molecular partners imparts significant in-droplet HDX protection for this peptide. In the future, a scale may be developed whereby HDX reactivity is predictive of the degree of structural flexibility and bias (propensity to form 2° structural elements such as α-helix, β-sheet, and β-turn) for intrinsically disordered regions (IDRs). Such structural resolution may ultimately be used for high-throughput screening of IDR structural transformation(s) upon binding of ligands such as drug candidates.

Figure 1

HDX behavior of the different model peptides. Scatter plots of analyte, PA (Panel A), PS (Panel C), BK (Panel B), and KDD (Panel D). Inset legends provide colors and symbols for charge states. Best-fit lines (dotted lines) are shown and were obtained with the LINEST function in Excel. See Table S1 in the Supporting Information section.

Figure 2

Relationship between theoretical and experimental exchange values for the various models described in the text. Panel A shows the experimental % BB exchange as a function of total backbone exchangeable sites for all peptides. Panel B shows the experimental % BB exchange as a function of the theoretical % BB exchange for the M_intra (red symbols) and M_inter (black symbols) models for all peptides. Panel C shows the relationship between experimental % BB exchange and theoretical % BB exchange for the M_CMB model. Dotted lines show the best-fit lines obtained from the LINEST function (see Table 2). Individual peptides are labeled in each panel. y-axis error bars (measurement uncertainties) are obtained by propagation of error and x-axis error bars represent standard error of the meant. These calculations are provided in the Error Calculation section of the Supporting Information.

Figure 3

HDX analysis as described by PF_i for the model peptides. For M_inter and M_intra models, panels A and B, respectively, show the ln(PF_i) data for PS, BK, Nt17, PA, and KDD. Panels C and D (respective models) show the per-residue HDX contributions for the same peptides using the M_CMB approach with the inter- (panel C) and intramolecular (panel D), HB treatment, respectively. To obtain these HDX contribution values, eq 6 was used (see text for details). Error bars represent the standard error of the mean from multiple (n ≥ 3) replicates (see Error Calculation section in the Supporting Information).

Figure 4

Secondary structure analysis of Nt17. Structural evolution plots including: Panel A showing helical content, Panel C showing a 2D histogram of residue (i) vs residue (j) contact distribution, Panel B showing a density plot of backbone root-mean-square deviation (RMSD), and Panel D showing the radius of gyration (R_g) vs Asphericity (δ). In Panel C, the Frequency (unitless) color bar represents the degree of an event (i and j coming within HB contact distance). The Density (unitless) in Panel C refers to the fraction of conformers having the RMSD value and r1, r2, r3 refer to 3 separate MD trajectories. For results from replicate MD simulations, see Figures S12–S14.