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Abstract
The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. 1H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, 3JαH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.
| Abbreviations | ||
| CD | = | circular dichroism |
| CSI | = | chemical shift index |
| DIPEA | = | di-iso-propylethylamine |
| DMF | = | N,N-dimethylformammide |
| DSS | = | 4,4-dimethyl-4-silapentane-1-sulfonate |
| EDT | = | 1,2-ethanedithiol |
| Fmoc | = | 9-fluorenylmethyloxycarbonyl |
| HPLC | = | high performance liquid chromatography |
| NMR | = | nuclear magnetic resonance |
| NOE | = | nuclear Overhauser effect |
| OGC | = | oxoglutarate carrier |
| PyBop | = | benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophoshate |
| RMSD | = | root-mean-square deviation |
| SDS | = | sodium dodecyl sulphate |
| TCEP | = | tris (2-carboxyethyl)phosphine |
| TFA | = | trifluoroacetic acid |
| TFE | = | trifluoroethanol |
| TM | = | transmembrane |
| TMS | = | transmembrane segment |
| Abbreviations | ||
| CD | = | circular dichroism |
| CSI | = | chemical shift index |
| DIPEA | = | di-iso-propylethylamine |
| DMF | = | N,N-dimethylformammide |
| DSS | = | 4,4-dimethyl-4-silapentane-1-sulfonate |
| EDT | = | 1,2-ethanedithiol |
| Fmoc | = | 9-fluorenylmethyloxycarbonyl |
| HPLC | = | high performance liquid chromatography |
| NMR | = | nuclear magnetic resonance |
| NOE | = | nuclear Overhauser effect |
| OGC | = | oxoglutarate carrier |
| PyBop | = | benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophoshate |
| RMSD | = | root-mean-square deviation |
| SDS | = | sodium dodecyl sulphate |
| TCEP | = | tris (2-carboxyethyl)phosphine |
| TFA | = | trifluoroacetic acid |
| TFE | = | trifluoroethanol |
| TM | = | transmembrane |
| TMS | = | transmembrane segment |