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. 2018 Oct 18;7:e40110. doi: 10.7554/eLife.40110

Figure 2. | Deuterium exchange at 20°C.

(a-c) Deuterium uptake kinetics at 20°C of examples peptides from EAAT1WT (grey), EAAT1CO (blue), and EAAT1COCO (red), respectively, covering both helical and unstructured regions of the transporters. Solid lines represent double-exponential fits to the data, and dotted lines the expected deuterium kinetics of unfolded and solvent exposed peptides (see ‘HDX kinetic analysis’ in Methods). Plots in (a–c) depict an average of three independent experiments, and error bars represent s.e.m. (d-f) Deuterium incorporation after 1 hr at 20°C in EAAT1CO (d), EAAT1COCO (e), and EAAT1WT (f) mapped into the structure of the EAAT1CRYTS (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, and black lines indicate the approximate position of some peptides in the structure, and the substrate (Asp). In the trimeric depiction, arrows point to the interface between protomers. Deuterium incorporation was calculated as an average of three independent experiments, and normalized to the maximal theoretical incorporation based on the number of backbone amide available for exchange in each peptide. The color code representing deuterium incorporation is depicted in a scale bar (d).

Figure 2.

Figure 2—figure supplement 1. | Deuterium exchange behavior of EAAT1CO.

Figure 2—figure supplement 1.

(a) Sequence coverage of EAAT1COCO obtained after 2 min pepsin digestion. Each bar below the protein sequence corresponds to a unique peptide identified by data independent MS/MS acquisition. A total of 49 peptides (colored in blue) covering 69.5% of the EAAT1CO sequence were selected for data extraction. Two extra N-terminal residues (GP) remaining after PreScission cleavage are also reported. (b) HDX-MS profile of EAAT1CO. The relative fractional uptake values determined for each peptide and at each time point are plotted as a function of peptide position in the protein sequence. The color code of the MS data acquired from 10 s up to 1 hr is indicated in the upper part of the panel. Each point corresponds to the average fractional uptake value obtained from three independent replicates. Small color bars indicate helical regions of the TranD (orange) and ScaD (blue).
Figure 2—figure supplement 2. | Deuterium exchange behavior of EATTCOCO.

Figure 2—figure supplement 2.

(a) Sequence coverage of EAAT1COCO obtained after 2 min pepsin digestion. Each bar below the protein sequence corresponds to a unique peptide identified by data independent MS/MS acquisition. A total of 53 peptides (colored in blue) covering 78.3% of the EAAT1COCO sequence were selected for data extraction. The two extra N-terminal residues (GP) remaining after PreScission cleavage are also reported. (b) HDX-MS profile of EAAT1COCO. The relative fractional uptake values determined for each peptide and at each time point are plotted as a function of peptide position in the protein sequence. The color code of the MS data acquired from 10 s up to 1 hr is indicated in the upper part of the panel. Each point corresponds to the average fractional uptake value obtained from three independent replicates. Small color bars indicate the helical region of the TranD (orange) and ScaD (blue).
Figure 2—figure supplement 3. | Examples of representative unimodal m/z envelopes (EX2 kinetics) observed in EAAT1CO and EAAT1COCO.

Figure 2—figure supplement 3.

(a-b) Raw MS data of example peptides 357–369 (a) and 390–399 (b) in EAAT1CO (left column) and EAAT1COCO (right column). The top mass spectra represent the isotopic distribution of the undeuterated (UND) peptides. One unique isotopic distribution is observed during the time course of the experiment indicative of EX2 kinetic.
Figure 2—figure supplement 4. | Deuterium uptake kinetics in EAAT1WT, EAAT1CO, and EAAT1COCO.

Figure 2—figure supplement 4.

Deuterium uptake kinetic plots at 20°C of example peptides from EAAT1WT (grey), EAAT1CO (blue), and EAAT1COCO (red), respectively, covering both helical and unstructured regions of the transporters. Solid lines represent double exponential fits to the data, and dotted lines the expected deuterium kinetics of unfolded and solvent exposed peptides (see methods). Labels at the top of the plots indicate the positions of residues in the sequence as well as the regions of the transporter covered by the peptides and their involvement in substrate (subst.) and sodium 1 – 3 (Na1-3) coordination (in brackets), respectively. Plots depict an average of three independent experiments, and error bars represent s.e.m.
Figure 2—figure supplement 5. | Deuterium exchange behavior of EAAT1WT.

Figure 2—figure supplement 5.

(a) Sequence coverage of EAAT1WT obtained after 2 min pepsin digestion. Each bar below the protein sequence corresponds to a unique peptide identified by data independent MS/MS acquisition. A total of 48 peptides (colored in blue) covering 74.8% of the EAAT1WT sequence were selected for data extraction. The two extra N-terminal residues (GP) remaining after PreScission cleavage are also reported. (b) HDX-MS profile of EAAT1WT. The relative fractional uptake values determined for each peptide and at each time point are plotted as a function of peptide position in the protein sequence. The color code of the MS data acquired from 10 s up to 1 hr is indicated in the upper part of the panel. Each point corresponds to the average fractional uptake value obtained from three independent replicates. Small color bars indicate the helical region of the TranD (orange) and ScaD (blue).