14 research outputs found

    FCS Study of the Thermodynamics of Membrane Protein Insertion into the Lipid Bilayer Chaperoned by Fluorinated Surfactants

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    Experimental determination of the free energy (ΔG) stabilizing the structure of membrane proteins (MPs) in their native environment has been hampered by the aggregation and precipitation of MPs outside the lipid bilayer. We recently demonstrated that the latter process can be prevented by the use of fluorinated surfactants, FTACs, that act as chaperones for MP insertion without partitioning in the membrane themselves. Here we combine the advantages of the chaperone-like ability of FTACs with the sensitivity of fluorescence correlation spectroscopy measurements to determine ΔG of bilayer insertion of model MPs. First, we calibrate our approach by examining the effects of chaperoned insertion on ΔG of transmembrane insertion of Annexin B12. We find that a shorter-chained surfactant, FTAC-C6, for which the working concentration range of 0.05–0.2 mM falls below CMC = 0.33 mM, has a mild effect on an apparent ΔG. In contrast, additions of a longer-chained FTAC-C8 (CMC = 0.03 mM) result in a steep and nonlinear concentration dependence of ΔG. We then apply the same methodology to the pH-triggered insertion of diphtheria toxin T-domain, which is known to be affected by nonproductive aggregation in solution. We find that the correction of the ΔG value needed to compensate for unchaperoned insertion of the T-domain exceeds 3 kcal/mole. A relatively shallow and linear dependence of the ΔG for Annexin B12 and T-domain insertion on FTAC-C6 concentration is encouraging for future applications of this surfactant in thermodynamic studies of the stability of other MPs

    Is Lipid Bilayer Binding a Common Property of Inhibitor Cysteine Knot Ion-Channel Blockers?

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    Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (ΔG) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of ΔG dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep ΔG dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed ΔG shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels
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