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. 2017 Dec 5;113(11):2311-2315.
doi: 10.1016/j.bpj.2017.09.019. Epub 2017 Oct 20.

Mitochondrial ADP/ATP Carrier in Dodecylphosphocholine Binds Cardiolipins With Non-native Affinity

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Free PMC article

Mitochondrial ADP/ATP Carrier in Dodecylphosphocholine Binds Cardiolipins With Non-native Affinity

François Dehez et al. Biophys J. .
Free PMC article

Abstract

Biophysical investigation of membrane proteins generally requires their extraction from native sources using detergents, a step that can lead, possibly irreversibly, to protein denaturation. The propensity of dodecylphosphocholine (DPC), a detergent widely utilized in NMR studies of membrane proteins, to distort their structure has been the subject of much controversy. It has been recently proposed that the binding specificity of the yeast mitochondrial ADP/ATP carrier (yAAC3) toward cardiolipins is preserved in DPC, thereby suggesting that DPC is a suitable environment in which to study membrane proteins. In this communication, we used all-atom molecular dynamics simulations to investigate the specific binding of cardiolipins to yAAC3. Our data demonstrate that the interaction interface observed in a native-like environment differs markedly from that inferred from an NMR investigation in DPC, implying that in this detergent, the protein structure is distorted. We further investigated yAAC3 solubilized in DPC and in the milder dodecylmaltoside with thermal-shift assays. The loss of thermal transition observed in DPC confirms that the protein is no longer properly folded in this environment.

Figures

Figure 1
Figure 1
Snapshot of yAAC3 after ∼150 ns of a 200 ns MD simulation. The membrane carrier is represented as a transparent solvent-accessible surface with its secondary structure inside. The amide sites for which NOE crosspeaks to CL atoms were observed (16), namely, I14, I100, G118, G127, F220, L282, S292, Q298, M299, and I300, are highlighted as red van der Waals spheres. The three CLs binding yAAC3 are shown in yellow and pink to distinguish the region of the protein being examined. To see this figure in color, go online.
Figure 2
Figure 2
Distance root mean-square deviation (RMSD) from the starting configuration computed over the heavy atoms of yAAC3 (A), the α-helical content of yAAC3 (B), the acyl-chain heavy atoms of the three CLs binding the mitochondrial carrier (C), and the heavy atoms of the CL headgroups (D). The instantaneous values of the RMSD are shown in gray and the running average in black.
Figure 3
Figure 3
Distance separating the –NH hydrogen atom of residues I14 (A) and L282 (B) from the four terminal methyl groups of the closest CL. The four distances are shown as cyan, turquoise, light green, and dark green curves, alongside their mean, represented by a thick gray curve, and the running average thereof, represented as a black curve. Probability distributions of the four aforementioned hydrogen-methyl distances for residues I14 (C) and L282 (D). To see this figure in color, go online.
Figure 4
Figure 4
Thermostability of yAAC3 in 0.1% DDM (blue line) or 0.1% DPC (red line), in the presence (dashed line) and absence (straight line) of CATR. Thermostability was monitored using (A) the maleimide coumarin fluorophore CPM (24) and (B) nanoDSF (see Supporting Material). The top panels show the changes in fluorescence with temperature, whereas the bottom panels show the derivatives and the apparent melting temperatures, where they could be determined. To see this figure in color, go online.

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