Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes

Abstract

Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.

Keywords: Binding affinity; Differential scanning fluorimetry; Kinetic stability; MHC protein; Thermal stability.

Figure 1

Quantitative analysis of differential scanning fluorimetry in assessing class I and class II MHC/peptide stability. A) Progress curves for different concentrations of the class I TYR/A2 complex. Overall fluorescent intensity varied with concentration, but the midpoint of the transition was a constant 67 °C, as indicated by the vertical dashed line. B) The first derivative of the progress curves provides a means to robustly determine T_m values, as shown for the TYR/A2 complex at 2 µM. Black dots indicate the first derivative data; the red line is a baseline-corrected bi-Gaussian peak fit. C) First-derivative analysis and associated fit of the data for the class II HA/DR1 complex (circles) and ‘empty’ DR1 (triangles). D) peptide/MHC T_m values determined by DSF correlate with those determined by CD spectroscopy. Black circles are class I complexes; blue triangles are class II. The correlation coefficient (R) for the linear fit to the data (red line) is 0.98. E) peptide/A2 denaturation remains irreversible when studied by DSF. The black curve shows the DSF scan of a fresh sample of the SL9 peptide bound to A2 with a T_m of 59 °C. The purple line shows a rescan of the same sample after cooling to 25 °C. F) Thermal denaturation of peptide/A2 complexes show a scan rate dependence, as indicated here for the MART1_26(2L)-35 peptide bound to A2.

Figure 2

T_m values determined by DSF correlate with predicted and measured IC₅₀ values. A) Comparison of DSF-determined T_m values and IC₅₀ values predicted by NetMHC and NetMHCII. Black circles are class I complexes; blue triangles are class II. The solid red line is a linear fit to the class I and class II data together. The upper dashed line is a linear fit to the class II data alone, and the lower dashed line is a linear fit to the class I data alone. Correlation coefficients for all three fits are indicated. B) Comparison of DSF-determined T_m values and experimental IC₅₀ values for peptide/DR1 complexes. The red line is a linear fit to the data. The correlation coefficient is 0.85.

Figure 3

DSF detects separate β₂m unfolding in low stability class I MHC complexes. A) Derivative analyses of the progress curves for the MART-1_27-35 and HUD complexes with A2. A separate peak is seen for both with a T_m at or near 63 °C. The pink dashed line shows the fit from an analysis of β₂m alone superimposed on the MART-1 and HUD data. B) DSF analysis of β₂m alone shows a single transition with a T_m of 63 °C. The fitted curve from this analysis is shown as the pink dashed line in panel A. The reduced intensity of the β₂m alone sample in panel B compared to the pMHC samples in panel A is attributable to a lower concentration and the smaller size of β₂m, resulting in less binding SYPRO orange.

Figure 4

Differential scanning fluorimetry can be used to assess peptide dissociation kinetics and thus the kinetic stability of class I MHC complexes. A) Dissociation of the native MART-1_27-35 nonamer (circles) and anchor-modified variant (triangles) from HLA-A2 at 37 °C. B) Dissociation of the native MART-1_26-35 decamer (circles) and anchor-modified variant (triangles) from HLA-A2 at 37 °C. For both panels, red lines indicate biexponential fits to the data. The slowest time constant was used to report complex half-lives, indicated in the inset as _t1/2. Half-lives for the faster time constants in hours are 0.4 for AAGIGILTV, 0.5 hours for ALGIGILTV, 0.3 hours for EAAGIGILTV, and 1.1 hours for ELAGIGILTV.