Screening of candidate substrates and coupling ions of transporters by thermostability shift assays
- University of Cambridge, United Kingdom
- Macquarie University, Australia
- University of Leeds, United Kingdom
Figures
Figure 1 with 1 supplement
Substrate-induced stabilization of a mitochondrial ADP/ATP carrier.
(a) As protein molecules in a population unfold due to a gradual rise in temperature (25–90°C), buried cysteine residues become solvent-exposed and accessible to the thiol-specific probe CPM (blue …
Figure 1—figure supplement 1
Substrate-induced stabilization of a mitochondrial ADP/ATP carrier.
The substrate concentration-dependency of the apparent melting temperature of purified mitochondrial ADP/ATP carrier. The apparent melting temperature (Tm) was determined from the peak in the …
Figure 2 with 4 supplements
Validation of the method for determining substrate specificity using three unrelated proteins.
(a), (c) and (e) Structural models of GalP (based on GLUT5, PDB 4YBQ), Mhp1 (PDB 2JLN) and AAC (based on Aac2p, PDB 4C9G), respectively. The models are shown in rainbow cartoon and wheat surface …
Figure 2—figure supplement 1
Substrate-induced stabilization of the galactose transporter GalP.
Typical thermostability profiles of purified GalP (top) and their derivatives (dF/dT; bottom) in the absence and presence of stabilizing substrates D-galactose (red trace), D-glucose (blue trace) …
Figure 2—figure supplement 2
Glucosamine is a transported substrate of the galactose transporter GalP.
Transport assays with [14C]-labeled D-glucosamine in whole cells of the GalP-expressing plasmid pGP1 in the host E. coli strain JM1100 (open circles) and control JM1100 without plasmid (closed …
Figure 2—figure supplement 3
Ligand-induced stabilization of Mhp1 in the presence of sodium ions.
Typical thermostability profiles of purified Mhp1 (top) and their derivatives (dF/dT; bottom) in the absence or presence of stabilizing compounds 5-benzyl-L-hydantoin (L-BH, red trace), …
Figure 2—figure supplement 4
Substrate screening of the mitochondrial ADP/ATP carrier.
Purified ADP/ATP carrier in dodecyl-maltoside was separately mixed with 2.5 mM of library compounds in 20 mM HEPES pH 8.0, 100 mM NaCl, 0.1% dodecyl-maltoside, 0.1 mg ml−1 tetraoleoyl cardiolipin …
Figure 3 with 3 supplements
Identification of substrates for a mitochondrial phosphate carrier from the thermophilic ciliate Tetrahymena thermophila.
Purified carrier (1 μg) in lauryl maltose neopentyl glycol was incubated at pH 6.0 with 2.5 mM of each library compound separately and the ΔTm was determined, which is the difference between the …
Figure 3—figure supplement 1
Substrate-induced stabilization of the mitochondrial phosphate carrier.
Typical thermostability curves of purified phosphate carrier (top) and their derivatives (dF/dT; bottom) in the absence and presence of stabilizing compounds phosphate (red trace), glyoxylate (blue …
Figure 3—figure supplement 2
Chemical structures of compounds that showed significant thermostability shifts with the mitochondrial phosphate carrier.
https://doi.org/10.7554/eLife.38821.011
Figure 3—figure supplement 3
Phosphate is transported by the putative phosphate carrier of Tetrahymena thermophila.
(A) Transport assays were initiated by the addition of 20 μM. [33P]-labeled orthophosphate in proteoliposomes with reconstituted carrier in the absence of internal orthophosphate (triangles) or the …
Figure 4
The effect of coupling ions on the stabilization of transporters by substrate binding.
(a) Thermostability shifts of Mhp1 (3 μg) induced by binding of hydantoins in the presence of 140 mM NaCl, KCl, CaCl2 and MgCl2. L-BH: 5-benzyl-L-hydantoin, BVH: 5-bromovinylhydantoin, L-IMH: …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | Dodecyl-β-D-maltoside | Glycon Biochemicals GmbH | Glycon:D97002 | |
| Chemical compound, drug | Dodecyl maltose neopentyl glycol | Anatrace | Anatrace:NG310 | |
| Chemical compound, drug | N-[4-(7-diethylamino-4-methyl-3- coumarinyl)phenyl] maleimide (CPM) | Sigma | Sigma:C1484 | |
| Chemical compound, drug | Complete Mini EDTA-free protease inhibitor tablets | Roche | Roche:5056489001 | |
| Chemical compound, drug | Nickel Sepharose (High Performance) | GE Healthcare | GE Healthcare:17526802 | |
| Chemical compound, drug | Nickel-NTA Superflow | Qiagen | Qiagen:30430 | |
| Chemical compound, drug | Factor Xa protease | NEB | NEB:P8010L | |
| Chemical compound, drug | Tetraoleoyl cardiolipin (TOCL) | Avanti Polar Lipids | Avanti Polar Lipids:710335C | |
| Chemical compound, drug | E. coli polar lipid extract | Avanti Polar Lipids | Avanti Polar Lipids:100600C | |
| Chemical compound, drug | [14C]-galactose | American Radiolabeled Chemicals | American Radiolabeled Chemicals:ARC0117 | |
| Chemical compound, drug | [14C]-glucosamine | American Radiolabeled Chemicals | American Radiolabeled Chemicals:ARC0118A | |
| Chemical compound, drug | [33P]-orthophosphate | Hartmann Analytic | Hartmann Analytic:FF-01 | |
| Chemical compound, drug | BioBeads | BioRad | BioRad:152–3920 | |
| Chemical compound, drug | C10E5 | Sigma | Sigma:76436 | |
| Chemical compound, drug | Hydantoin compounds | Other | A gift from Marta Sans, Maria Kokkinidou and Arwen Pearson, University of Hamburg | |
| Software, algorithm | Prism | GraphPad | ||
| Strain, strain background (S. cerevisiae) | Saccharomyces cerevisiae W303-1B | ATCC | ATTC:201238 | |
| Strain, strain background (S. cerevisiae) | Saccharomyces cerevisiae WB12 | PMID:9878703 | A gift from Dr H. Terada, Tokyo University of Science | |
| Strain, strain background (E. coli) | Escherichia coli JM1100 | PMID:15558 | ||
| Recombinant DNA reagent | Modified pYES3 vector | PMID:26453935 |
Additional files
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Supplementary file 1
Current status of identification of transport proteins in different archaeal, eubacterial and eukaryotic species (April 2018).
- https://doi.org/10.7554/eLife.38821.014
-
Supplementary file 2
Key resources table.
- https://doi.org/10.7554/eLife.38821.015
