Consensus designs and thermal stability determinants of a human glutamate transporter
- Institut Pasteur, France
- UMR 3528, CNRS, Institut Pasteur, France
- USR 2000, CNRS, Institut Pasteur, France
Figures
Figure 1 with 1 supplement
| EAAT1 consensus mutants.
(a-b) Residues exchanged for consensus amino acids in EAAT1CO (a) and EAAT1COCO (b) are mapped into the structure of the EAAT1CRYST (PDB 5LLM) trimer viewed from the extracellular medium (left …
Figure 1—figure supplement 1
| Amino acid alignment EAAT1 constructs.
(a), Amino acid sequences of EAAT1CO, EAAT1COCO, and EAAT1WT are compared. The rectangles above the sequences highlight the transmembrane helices observed in the crystal structure of EAAT1CRYST (PDB …
Figure 2 with 5 supplements
| 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 …
Figure 2—figure supplement 1
| Deuterium exchange behavior of EAAT1CO.
(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 …
Figure 2—figure supplement 2
| Deuterium exchange behavior of EATTCOCO.
(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 …
Figure 2—figure supplement 3
| Examples of representative unimodal m/z envelopes (EX2 kinetics) observed in EAAT1CO and EAAT1COCO.
(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 …
Figure 2—figure supplement 4
| Deuterium uptake kinetics in EAAT1WT, EAAT1CO, and EAAT1COCO.
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. …
Figure 2—figure supplement 5
| Deuterium exchange behavior of EAAT1WT.
(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 …
Figure 3 with 1 supplement
| Subunit dissociation by SEC.
(a-c) Size-exclusion chromatograms of purified EAAT1CO (a), EAAT1COCO (b), and the chimeric transporter EAAT1-ScaDCO-TranDCOCO (c), respectively, pre-heated at different temperatures. The …
Figure 3—figure supplement 1
| Neurotransmitter uptake by EAAT1COCO pre-heated at 55°C.
a, Representative SEC profiles of purified EAAT1COCO samples at 4°C (reference temperature) or pre-heated at 55°C. The profiles are also shown in Figure 3b. (b), Radioactive L-glutamate uptake in …
Figure 4 with 2 supplements
| Temperature-induced HDX changes.
(a-b) Changes in deuterium incorporation induced by pre-pulses at nearly the T50-SEC of EAAT1CO (50°C) (a), and EAAT1COCO (40°C) (b), respectively, measured after 1 hr at 20°C. The changes are …
Figure 4—figure supplement 1
| Temperature-induced changes in EAAT1CO HDX pattern.
(a-c) Difference in fractional deuterium uptake between the reference pre-pulse temperature (20°C) and 40°C (a), 50°C (b), or 65°C (c) plotted as a function of the peptide position in the protein …
Figure 4—figure supplement 2
| Temperature-induced changes in EAAT1COCO HDX pattern.
(a-b) Difference in fractional deuterium uptake between the reference pre-pulse temperature (20°C), and 40 (a) or 50°C (b) plotted as a function of the peptide position in the protein sequence. The …
Figure 5
| Temperature-induced bimodal m/z envelopes at the trimeric interface.
(a-b) m/z envelopes of an example peptide covering residues 174–184 of EAAT1CO (a) and EAAT1COCO (b), respectively, at different pre-pulse temperatures. Grey symbols represent the average of three …
Figure 6 with 1 supplement
| Local thermal unfolding.
(a-b) Deuterium uptake kinetics at different pre-pulse temperatures of example peptides containing residues 174–184 in EAAT1CO (a), and EAAT1COCO (b), respectively. Solid lines represent …
Figure 6—figure supplement 1
| Deuterium uptake kinetics in EAAT1CO and EAAT1COCO 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 …
Figure 7
| Thermal unfolding pathway.
Cartoon representation of the temperature-induced structural changes observed in the consensus designs. At the reference temperature the transporters are trimeric and have the substrate (sticks) …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | EAAT1WT | DNA2.0 synthetic | Uniprot P43003-1 | Codon optimized Homo sapiens |
| Cell line (Homo sapiens) | HEK293F | Thermo Fisher | R79007 | negative for mycoplasma |
| Transfected construct (Homo sapiens) | EAAT1CO | This paper | ||
| Transfected construct (Homo sapiens) | EAAT1COCO | This paper | ||
| Transfected construct (Homo sapiens) | EAAT1 ScaCO-TranDCOCO | This paper | ||
| Recombinant DNA reagent | pCDNA3.1 | Invitrogen, doi: 10.1038/nature22064 | ||
| Software, algorithm | FELIX 4.1.2 | Photon Technology International/Horiba | ||
| Software, algorithm | DynamX 3.0 | Waters | ||
| Software, algorithm | HX-Express2 | http://www.hxms.com/HXExpress | ||
| Software, algorithm | Sigma Plot 12 | Sysat Inc. | ||
| Software, algorithm | Excel | Microsoft |
Additional files
-
Supplementary file 1
Alignment animal SLC1 homologs.
- https://doi.org/10.7554/eLife.40110.019
-
Supplementary file 2
Alignment PFAM curated SLC1.
- https://doi.org/10.7554/eLife.40110.020
-
Transparent reporting form
- https://doi.org/10.7554/eLife.40110.021
