Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

Figures & data

Figure 1. CD spectra of peptide F215-R246 in: (a) TFE at 100% (x), 95% (-), 80% (Δ), 50% (□), 20% (---); (b) SDS 5% (Δ), 2% (---), 1% (x), 0.1% (□); and of peptide W279-K305 in (c): TFE 100% (x), 95% (-), 80% (Δ), 50% (□), 20% (---); (d) SDS 5% (Δ), 2% (---), 1% (x), 0.1% (□). The data were expressed in terms of [Θ]_R, the molar ellipticity per residue.

Figure 2. Summary of sequential and medium-range NOEs observed for peptide F215-R246 (a) and peptide W279-K305 (b) in 80% TFE at 25°C. The thickness of lines is related to the intensity of NOEs. Open bars indicate overlapping peaks. Asterisks under the amino acid one-letter codes indicate apparent ³J_αH-NH coupling constants of non-Gly residues <6.0 Hz. Chemical Shift Index (CSI) of αH protons are shown below the bar diagrams for each peptide. Negative values indicate a helical conformation.

Figure 3. Left: Superposition of the fifty best structures of peptides F215-R246 (a) and W279-K305 (c), best-fitted on backbone atoms of residues L220-S237 and P284-M303, respectively. Structures are oriented with the N-terminus on the top of the figure. For peptide W279-K305 the side chains of Pro residues are arrowed, and are also shown in magenta colour online. For (a) the RMSD values are 0.90±0.32 Å on backbone atoms and 1.69±0.41 Å on heavy atoms and for (c) 0.81±0.32 Å on backbone atoms and 1.97±0.37 Å on heavy atoms, respectively. Right: Ribbon representations of the mean structure of the bundles of peptides F215-R246 (b) and W279-K305 (d). This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 4. Quenching of intrinsic tryptophan fluorescence upon binding of TMSs to W279-K305. The spectra were recorded on a Fluorolog-3 spectrofluorimeter at an excitation wavelength of 295 nm. The fluorescence emission was recorded from 305 nm to 500 nm. (a) the spectra are W279-K305 alone (^_) and then in presence of peptides: K21-K46 (KSVKFLFGGLAGMGATVFVQPLDLVK) (Δ), R78-R108 (RGIYTGLSAGLLRQATYTTTRLGIYTVLFER) (x), P117-A149 (PGFLLKAVIGMTAGATGAFVGTPAEVALIRMTA) (□), F215-R246 (---); (b) W279-K305 alone (^_) and then in presence of 5 (---), 11 (□), 22 (Δ), 33 (•), 44 (x) and 55 (○) µM of F215-R246. The inset shows the fluorescence quenching of W279-K305 upon addition of different concentrations of F215-R246.

Figure 5. Summary of sequential and medium-range NOEs observed for peptide P257-L278 in 80% TFE at 25°C. The thickness of lines is related to the intensity of NOEs. Open bars indicate overlapping peaks. Asterisks under the amino acid one-letter codes indicate apparent ³J_αH-NH coupling constants of non-Gly residues <6.0 Hz. Chemical Shift Index (CSI) of αH protons are shown below the bar diagrams. Negative values indicate a helical conformation.

Figure 6. (a) Superposition of the fifty best structures of peptide P257-L278 best-fitted on backbone atoms of residues G262-F275. Structures are oriented with the N-terminus on the top of the figure. The RMSD values are 0.66±0.21 Å on backbone atoms and 1.93±0.36 Å on heavy atoms. (b) Ribbon representation of the mean structure of the bundle. This Figure is reproduced in colour in Molecular Membrane Biology online.

Figure 7. Helical wheel of peptides F215-R246, W279-K305 and P257-L278 obtained with the software ANTHEPROT (Deléage et al. [1988]). Hydrophobic residues are shown in blue, the hydrophilic residues in red, cysteine residues in green and the others in grey.

Figure 8. Superposition of backbone atoms of the mean structures of peptides F215-R246 (only residues L220-M238 are shown, in deep pink), W279-K305 (only P284-E301, in sky blue) and P257-L278 (only G262-E273, in yellow) on the corresponding amino acid sequences of helices H5 (residues 209–238), H6 (residues 273–290) and h56 (residues 253–264) in the crystal structure of the ADP/ATP carrier (Pebay-Peyroula et al. [2003]) (PDB accession code: 1okc, in dark blue). Only the region of the ADP/ATP carrier, which is relevant for the comparison with the peptides studied in this paper, is shown for clarity.

Deléage G, Clerc FF, Roux B, Gautheron DC. ANTHEPROT: A package for protein sequence analysis using a microcomputer. Comput Appl Biosci 1988; 4: 351–356

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Pebay-Peyroula CE, Dahou-Gonzales C, Kahn R, Trezeguet V, Lauquin GJM, Brandolin G. Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 2003; 426: 39–43

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