FCS study of the thermodynamics of membrane protein insertion into the lipid bilayer chaperoned by fluorinated surfactants.
Experimental determination of the free energy (DeltaG) 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 DeltaG of bilayer insertion of model MPs. First, we calibrate our approach by examining the effects of chaperoned insertion on DeltaG 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 DeltaG. In contrast, additions of a longer-chained FTAC-C8 (CMC = 0.03 mM) result in a steep and nonlinear concentration dependence of DeltaG. 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 DeltaG value needed to compensate for unchaperoned insertion of the T-domain exceeds 3 kcal/mole. A relatively shallow and linear dependence of the DeltaG 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.
Posokhov
Yevgen O
YO
Rodnin
Mykola V
MV
Das
Somes K
SK
Pucci
Bernard
B
Ladokhin
Alexey S
AS
D017302:Annexins / Q000378:metabolism*; D017611:Fluorine Compounds / Q000494:pharmacology*; D008051:Lipid Bilayers / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000494:pharmacology*; D013816:Thermodynamics; D017186:Titrimetry
D017302:Annexins; D017611:Fluorine Compounds; D008051:Lipid Bilayers; D008055:Lipids; D008565:Membrane Proteins; D013501:Surface-Active Agents