Figure 6. | Local thermal unfolding.

(a-b) Deuterium uptake kinetics at different pre-pulse temperatures of example peptides containing residues 174–184 in EAAT1_CO (a), and EAAT1_COCO (b), respectively. Solid lines represent double-exponential fits to the data, and dotted lines the expected deuterium kinetics of the unfolded and solvent exposed peptide at 20°C (see methods). Plots in (a–b) depict an average of three independent experiments, and error bars represent s.e.m. (c), Depiction of the temperature protocol used to prepare the protein samples. (d), The fraction of the slow HDX component extracted from the double-exponential fit to the kinetic data in (a) and (b) corresponding to EAAT1_CO (blue symbols) and EAAT1_COCO (red symbols), respectively. Lines represent the fit of a Hill-like equation with T_50-HDX-Uni values 51.9°C and 40.6°C in EAAT1_CO and EAAT1_COCO, respectively, and H values of −18.7 and −20, respectively. (e–f) T_50-HDX-Uni values, calculated as in (d), of EAAT1_CO (e) and EAAT1_COCO (f), respectively, are mapped into the structure of the EAAT1_CRYST (PDB 5LLM) trimer viewed from the extracellular medium (upper panel), as well as the scaffold (ScaD) and the transport (TranD) domains viewed from the membrane (lower panel), respectively. These domains are depicted separately for clarity of display. The color code representing T_50-HDX-Uni values is depicted in a scale bar between the trimers. Peptides that did not show any temperature-induced HDX changes are also labeled in cyan (T_50-HDX-Uni>60°C).

Figure 6—figure supplement 1. | Deuterium uptake kinetics in EAAT1_CO and EAAT1_COCO at different temperatures.

Deuterium uptake kinetic plots at different pre-pulse temperatures of example peptides covering different regions of the ScaD (peptides 162–170, and 187–194) and the TranD (peptides 374–389, and 397–404) in EAAT1_CO (left column), and EAAT1_COCO (middle column), respectively. The color code for the temperatures used in symbols and lines is indicated in the corresponding 162–170 peptide plots. Solid lines represent global double-exponential fits to the data obtained at all measured temperatures for an individual peptide (see methods). Dotted lines represent the expected deuterium kinetics of the unfolded and solvent exposed peptide at 20°C. Plots depict an average of three independent experiments, and error bars represent s.e.m. From the kinetic fits, the computed fraction of the slow HDX component is plotted as a function of temperature pre-pulses in EAAT1_CO (blue) and EAAT1_COCO (red symbols), respectively (right column). Solid and dashed lines represent the fit of a Hill-like equation (see methods) to EAAT1_CO and EAAT1_COCO data, respectively, with T_50-HDX-Uni values depicted in the plots.