COLL 588 |
| Fluorinated (e.g. F-TAC) and hemifluorinated compounds (e.g. HF-TAC) are a novel class of non-detergent surfactants designed to keep membrane proteins soluble under non-dissociating conditions. Because fluorinated and hydrogenated chains do not mix well, supramicellar concentrations of these surfactants are expected to cause minimal membrane perturbation while ensuring solubility of membrane proteins. To test the ability of (H)F-TAC to assist proper membrane insertion of proteins, we examined its effect on the pH-triggered insertion of diphtheria toxin T-domain. The function of the T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome. This translocation is accompanied by the formation of a pore, which we used as a measure of activity in a vesicle leakage assay. We have also used FRET to follow the effect of (H)F-TAC on aggregation of aqueous and membrane-bound T-domain. We characterized membrane interactions of the T-domain in the presence of surfactants by means of fluorescence correlation spectroscopy. Our data indicate that the pore-forming activity of the T-domain is affected by the dynamic interplay of two principal processes: productive pH-triggered membrane insertion and nonproductive aggregation of aqueous at low pH. Addition of fluorinated surfactants to the buffer prior to acidification is demonstrated to suppress aggregation in solution and ensure correct insertion/folding of the T-domain into the lipid bilayer. This chaperone-like activity of fluorinated surfactants opens up the possibility for their use in thermodynamic characterization of membrane folding/insertion of both constitutive and non-constitutive membrane proteins. Supported by NIH grants GM-069783, RR-016443. |
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Chemistry at Membrane Interfaces
8:30 AM-11:50 AM, Thursday, 14 September 2006 Sir Francis Drake -- Carmel Room, Oral
Division of Colloid & Surface Chemistry |